EP0937044B1 - Cftr channel activator compounds and pharmaceutical compositions containing same - Google Patents

Description

The present invention relates to compounds activating the channel CFTR, pharmaceutical compositions containing these, as well as their use in the treatment of pathologies such as cystic fibrosis.

In an epithelial cell, the transport of water and electrolytes are associated with an increase in membrane permeabilities for the K ⁺ , Na ⁺ and Cl ^- ions. These movements are linked to the activity of ion channels, namely specialized proteins, integrated into the membrane allowing the passive diffusion of ions. Molecular electrophysiology techniques (patch clamp) allow the recording at the unit level of the openings and closings of an ion channel and make possible the study of transepithelial ion transportations, their regulations and their pathological disturbances.

Among the many pathologies associated with cell physiology epithelial, cystic fibrosis is also considered a pathology ion channels insofar as the protein involved is a channel chloride, the CFTR channel for "Cystic Fibrosis Transmembrane conductance Regulator ". Cystic Fibrosis (CF) in the terminology Anglo-Saxon is the most common autosomal recessive genetic disorder in Caucasian populations. In the United States and most countries in Europe, the frequency of carriers of the CF gene is 1 in 20 to 1 in 30. The Cystic fibrosis affects the exocrine glands of the human body. The main expression sites of the CFTR protein are the exocrine pancreas, lungs, sweat glands, gut and heart tissue. Interest to this disease had important consequences for the understanding of secretory mechanisms of normal epithelial cells. Cells epithelial cells of the exocrine glands of the intestine, pancreas or lungs control the transport of salt and water in these organs. In cystic fibrosis, mutations in the CF gene alter the properties and function of the CFTR channel. The electrolytic transport then becomes abnormal and leads to disorders chronic obstructive pulmonary disease, pancreatic insufficiency, bacterial lung disease, abnormally sweat secretion concentrated and male infertility. The secretion defect is linked to operation of selective ion channels for chloride ions (channels CFTR), located in the apical membrane of cells and whose activity is controlled by the cyclic AMP route.

The CFTR protein is a glycoprotein of 1480 amino acids, with a weight molecular mass of 170 kD divided into five domains (Riordan et al., 1989), two transmembrane segments with each 6 alpha helices (numbered from 1 to 12 each with 21 to 22 amino acids), two domains of binding nucleotides (NBF1 and 2) and a broad hydrophilic regulatory domain (domain R). The CFTR protein, by its molecular structure belongs to the family membrane transporters (ABC for ATP-binding cassette).

ABC transporters are a family of membrane proteins very preserved in evolution. They are involved in the translocation of Various substrates across cell membranes. However, while at the prokaryotes of many carrier / substrate pairs have been defined, these information is more scarce in eukaryotes. In mammals, the most ABC transporters are associated with pathology. Let's quote protein CFTR involved in cystic fibrosis, P glycoprotein (MDR; Multi Drug Resistance) involved in the rejection of cytotoxic anti-tumor drugs and the newly described protein ABC1 which plays an essential role in endocytosis of apoptotic bodies by the macrophage. The CFTR controls the transport of transepithelial chloride and hydration of the compartments mucosal, while one of the MDR isoforms is involved in the translocation of phosphatidylcholine. These three ABC proteins that have a structure with twice 6 transmembrane segments, have two domains that bind and hydrolyze nucleotides (NBF) and a domain regulator. The regulation of CFTR has been particularly studied.

Two complex processes control the activity of the CFTR channel: phosphorylation of the R domain by protein kinases and fixation (and perhaps hydrolysis) of ATP on the two NBF domains. The dephosphorylation of CFTR channel causes a loss of activity of the channel until its closure (Tabcharani et al., 1991, Becq et al., 1993a, Becq et al., 1994). In addition, the CFTR channel is associated with a membrane phosphatase which controls the activity and the state of channel phosphorylation (Becq et al., 1993b, Becq et al., 1994).

The gene coding for the protein CFTR, was isolated by molecular cloning and identified on chromosome 7 (Kerem et al., 1989, Riordan et al., 1989). The identification of the gene and its implication in cystic fibrosis have been confirmed by the location of a deletion of three base pairs in a coding region (exon 10) of the CF gene from CF patients. This mutation corresponds to the deletion of a phenylalanine in position 508 (ΔF508) of the protein in NBFI. The frequency of occurrence of this mutation is 70% on average in genetic analyzes (Tsui & Buchwald, 1991). The consequences of this mutation are dramatic because the abnormal protein from of the transcription of the mutated gene (ΔF508) is no longer capable of ensuring its function in the chloride transport of affected epithelial cells. The absence of chlorine current after stimulation of the epithelial cells of cAMP exocrine glands is the main feature showing the presence of an abnormality in the CF gene and in particular of the mutation (ΔF508). More than 300 mutations have been identified to date on the CF gene. The density of highest mutations are found in the two areas of fixation of nucleotides. The mutation (ΔF508) is found in 70% of cases and 50% of patients are homozygous for this mutation. Seven other mutations important are present with frequencies higher than 1%. The mutation G551D corresponds to the substitution of a glycine residue (G) in position 551 of protein with aspartic acid (D). Patients with this mutant have severe pathology with pancreatic insufficiency and disorders severe lung disease (Cutting et al., 1990). The frequency of observation and this mutation reaches 3 to 5% in certain CF populations. Unlike deletion ΔF508, the CFTR protein carrying the G551D mutation is mature and is incorporated into the membrane (Gregory et al., 1991). However, the mutation results in membrane impermeability and stimulation of the cAMP pathway does not open the channel associated with the expression of this mutant (Gregory et al., 1991, Becq et al., 1994).

Other mutations like R117H, R334W and R347P appear with low frequencies of 0.8, 0.4 and 0.5% respectively and are associated with a less serious pathology (Sheppard et al., 1993). The expression of these three mutants generate a mature glycosylated form of the protein consistent with its insertion into the membrane.

The three mutants are capable, however, of responding to stimulation from the path of AMP by opening channels. The amplitude of the currents, the unit conductance and the probability of opening the channel associated with each of the three mutants are modified from the normal CFTR channel (Sheppard and al., 1993, Becq et al., 1994). Regulation by kinases / phosphatases seems however normal for these different mutants, including the G551D and ΔF508 (Becq et al., 1994).

Thus, these observations show that it is pharmacologically possible activate a large number of CFTR mutants, including G551D and ΔF508. Despite a lack of addressing of the ΔF508 protein in the membranes of epithelial cells affected by cystic fibrosis several groups have shown that this protein could be functionally present in membranes (Dalemans et al., 1991, Drumm et al., 1991, Becq et al., 1994). Thus, it appears necessary and essential to develop a strategy of CFTR channel openers to optimize the chances of success of a therapy but also to replace gene therapy when it is not necessary (mutations other than ΔF508).

Despite advances in the genetics of cystic fibrosis, the biology and biochemistry of the CFTR protein, the pharmacology of the CFTR channel is poorly developed. Three families of molecules are today put forward for their properties of activators or openers of the CFTR channel; phenylimidazothiazoles (levamisole and bromotetramisole), benzimidazolones (NSOO4) and xanthines (IBMX, theophylline ...).

1) phenylimidazothiazoles (levamisole and bromotetramisole)

It has recently been shown that levamisole and bromotetramisole make it possible, by inhibiting a membrane phosphatase, to control the activity and the level of phosphorylation of the CFTR channel (Becq et al., 1994,). These compounds open the CFTR channel in a dose-dependent manner (Becq et al 1996) and act on the CFTR channel presenting mutations which cause the disease (Becq et al., 1994). The mode of action of these activators is still uncertain. These molecules do not act by the conventional cAMP or intracellular calcium pathways. The compounds of the bromotetramisole family already have a therapeutic use (Grem, 1990) and levamisole is used in certain pulmonary therapies (Van Eygen et al., 1976, Dils, 1979). These latter properties represent a definite advantage for initiating clinical tests. However, these molecules do not seem to be able to act in all cells. The intestinal cells respond poorly and the opening of the CFTR after expression in the Xenope oocyte cannot be triggered. In addition, in a transgenic mouse model with the G551D / G551D mutation, bromotetramisole does not have the expected activating effect. The effects of these molecules therefore seem limited.

2) Benzimidazolones (NSOO4)

Recently, Gribkoff et al., 1994 showed that NS004, a compound (benzimidazolone) derived from the imidazole nucleus like levamisole can in certain conditions (when the CFTR has been phosphorylated) open the channel. The benzimidazolones are however also activators of many channels potassium (Olesen et al., 1994) and are therefore not very specific for CFTR.

3) Xanthines (IBMX, theophylline ...).

Xanthines such as IBMX (3-isobutyl-1-methylxanthine) are CFIR activators. The mechanism of action is still poorly understood and several possibilities exist. Xanthines can by inhibiting phosphodiesterases intracellular (cAMP degradation enzymes) increase the rate of cAMP and therefore activate CFTR. Other possibilities are advanced today like binding of xanthines to the binding sites for nucleotides (NBF) of CFTR.

The object of the present invention is to provide compounds which activate the CFTR channel which are more specific to CFTR than compounds CFTR channel activators described so far.

As such, the present invention aims to provide new drugs intended for the treatment of pathologies associated with disorders of the transmembrane ion fluxes, especially chlorine, in cells epithelial from a human or animal organism.

The object of the present invention is more particularly to provide new drugs that may be used to treat cystic fibrosis, prevention of rejection of cytotoxic drugs (including anti-tumor), or the prevention or treatment of obstructed pathways bronchial or digestive tract (including pancreatic or intestinal) or again as part of the treatment of cardiovascular disease.

Another object of the present invention is to provide a method of preparation of the compounds and pharmaceutical compositions of the invention.

   dans laquelle :

• l'hétérocycle A est aromatique ou non, étant entendu que dans ce dernier cas l'atome d'azote de cet hétérocycle est lié par une double liaison au carbone en position 4a,
• R₁, R₂, R₃, R₄, R₅, R₇, R₈, R₉ et R₁₀, représentent, indépendamment les uns des autres :
  • un atome d'hydrogène, ou de brome, ou de fluor, ou
  • un atome d'halogène, notamment un atome de chlore, ou
  • un groupe alkyle, alkoxy, carbonyle, ou oxycarbonyle, linéaire ou ramifié, d'environ 1 à environ 10 atomes de carbone, ces groupes étant le cas échéant substitués, notamment par un halogène, et/ou par un hydroxyle, et/ou par une amine (primaire, secondaire ou tertiaire), et/ou par un cycle aromatique et/ou aliphatique, d'environ 5 à environ 10 atomes de carbone dans le cycle, ces cycles étant eux-mêmes, le cas échéant, substitués notamment par un halogène, et/ou par un hydroxyle, et/ou par une amine (primaire, secondaire ou tertiaire), et/ou par un groupe alkyle, alkoxy, carbonyle, ou oxycarbonyle, ces groupes étant tels que définis ci-dessus, ou
  • un cycle aromatique ou aliphatique, d'environ 5 à environ 10 atomes de carbone dans le cycle, ce cycle étant lui-même, le cas échéant, substitué notamment par un halogène, et/ou par un hydroxyle, et/ou par une amine (primaire, secondaire ou tertiaire), et/ou par un groupe alkyle, alkoxy, carbonyle, ou oxycarbonyle, ces groupes étant tels que définis ci-dessus, ou
  • un groupe -OR_a, R_a représentant un atome d'hydrogène, ou un groupe alkyle, carbonyle, ou oxycarbonyle, linéaire ou ramifié, ces groupes étant tels que définis ci-dessus, ou un cycle aromatique ou aliphatique, ces cycles étant tels que définis ci-dessus, ou
  • un groupe -NR_bR_c, R_b et R_c, indépendamment l'un de l'autre, représentant un groupe alkyle, alkoxy, carbonyle, ou oxycarbonyle, linéaire ou ramifié, ces groupes étant tels que définis ci-dessus, ou un cycle aromatique ou aliphatique, ces cycles étant tels que définis ci-dessus, ou
  • lorsque R₁ et R₂, ou R₃ et R₄, et/ou R₄ et R₅, et/ou R₇ et R₈, et/ou R₈ et R₉, et/ou R₉ et R₁₀, ne représentent pas les différents atomes ou groupes ou cycles mentionnés ci-dessus, alors R₁ en association avec R₂, ou R₂ en association avec R₃, et/ou R₃ en association avec R₄, et/ou R₄ en association avec R₅, et/ou R₇ en association avec R₈, et/ou R₈ en association avec R₉, et/ou R₉ en association avec R₁₀, forment respectivement avec C₁ et C₂, ou avec C₂ et C₃, ou avec C₃ et C₄, ou avec C₄, C_4a et C₅, ou avec C₇ et C₈, ou avec C₈ et C₉, ou avec C₉ et C₁₀, un cycle aromatique ou aliphatique de 5 à 10 atomes de carbone, ce cycle étant le cas échéant substitué, notamment par un halogène, et/ou par un groupe alkyle, alkoxy, carbonyle, ou oxycarbonyle, et/ou par un cycle aromatique ou aliphatique, ces groupes ou cycles étant tels que définis ci-dessus, ou
  • lorsque R₃ et R₄ ne représentent pas les différents atomes ou groupes ou cycles mentionnés ci-dessus, alors R₃ en association avec R₄ forment un groupe indole de formule
    

   dans laquelle R_a est tel que défini ci-dessus,

• Y représente :
  • un groupe -OR_d, R_d représentant un atome d'hydrogène, ou un groupe alkyle, carbonyle, ou oxycarbonyle, linéaire ou ramifié, ces groupes étant tels que définis ci-dessus, ou un cycle aromatique ou aliphatique, ces cycles étant tels que définis ci-dessus, ou
  • un groupe -NR_eR_f, R_e et R_f indépendamment l'un de l'autre, représentant un groupe alkyle, alkoxy, carbonyle, ou oxycarbonyle, linéaire ou ramifié, ces groupes étant tels que définis ci-dessus, ou un cycle aromatique ou aliphatique, ces cycles étant tels que définis ci-dessus,
  • étant entendu que lorsque R_d, ou l'un au moins de R_e ou de R_f, ne représentent pas l'un des différents atomes ou groupes ou cycles mentionnés ci-dessus, alors R_d, ou l'un au moins de R_e ou de R_f, en association avec R₅, ou en association avec R₇, forment respectivement avec C₅ et C₆, ou avec C₆, C_6a et C₇, un hétérocycle aromatique ou aliphatique de 5 à 10 atomes de carbone, le cas échéant substitué, notamment par un halogène, et/ou par un groupe alkyle, alkoxy, carbonyle, ou oxycarbonyle, et/ou par un cycle aromatique ou aliphatique, ces groupes ou cycles étant tels que définis ci-dessus,
  • X représente un atome sous forme anionique, tel qu'un atome d'halogène, notamment un atome de brome ou de chlore, ou un groupe d'atomes sous forme anionique, tel qu'un perchlorate, et l'azote de l'hétérocycle A de la formule (I) est sous forme quaternaire et est lié d'une part par liaison covalente au carbone en position 11, et, d'autre part, par liaison ionique à X défini ci-dessus,
  • étant entendu que lorsque R₁ et R₁₀ ne représentent pas l'un des différents atomes ou groupes ou cycles mentionnés ci-dessus, alors R₁ en association avec R₁₀ forment avec C₁, l'azote de l'hétérocycle A de la formule (I), C₁₁, et C₁₀, un hétérocycle aromatique ou aliphatique de 5 à 10 atomes de carbone, le cas échéant substitué, notamment par un halogène, et/ou par un groupe alkyle, alkoxy, carbonyle; ou oxycarbonyle, et/ou par un cycle aromatique ou aliphatique, ces groupes ou cycles étant tels que définis ci-dessus, pour la préparation de médicaments destinés au traitement de pathologies, notamment pulmonaires, digestives ou cardiaques, liées à des troubles des flux ioniques transmembranaires, notamment de chlore et, le cas échéant, de bicarbonate, dans l'organisme (humain ou animal), notamment pour la préparation de médicaments destinés au traitement de la mucoviscidose, ou à la prévention du rejet de drogues cytotoxiques (notamment antitumorales), ou au traitement des obstructions des voies bronchiques ou des voies digestives (notamment pancréatique ou intestinale)

The subject of the present invention is the use of compounds corresponding to benzo [c] quinolizisium derivatives of general formula (III) below:

in which :

• heterocycle A is aromatic or not, it being understood that in the latter case the nitrogen atom of this heterocycle is linked by a double bond to carbon in position 4a,
• R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₇ , R ₈ , R ₉ and R ₁₀ , represent, independently of each other:
  • a hydrogen, or bromine, or fluorine atom, or
  • a halogen atom, in particular a chlorine atom, or
  • a linear or branched alkyl, alkoxy, carbonyl or oxycarbonyl group of approximately 1 to approximately 10 carbon atoms, these groups being optionally substituted, in particular by a halogen, and / or by a hydroxyl, and / or by an amine (primary, secondary or tertiary), and / or by an aromatic and / or aliphatic ring, of approximately 5 to approximately 10 carbon atoms in the ring, these rings being themselves, if necessary, substituted in particular by a halogen, and / or by a hydroxyl, and / or by an amine (primary, secondary or tertiary), and / or by an alkyl, alkoxy, carbonyl, or oxycarbonyl group, these groups being as defined above, or
  • an aromatic or aliphatic ring, of about 5 to about 10 carbon atoms in the ring, this ring itself being, where appropriate, substituted in particular by a halogen, and / or by a hydroxyl, and / or by an amine (primary, secondary or tertiary), and / or by an alkyl, alkoxy, carbonyl, or oxycarbonyl group, these groups being as defined above, or
  • a group -OR _a , R _a representing a hydrogen atom, or an alkyl, carbonyl or oxycarbonyl group, linear or branched, these groups being as defined above, or an aromatic or aliphatic ring, these cycles being such as defined above, or
  • a group -NR _b R _c , R _b and R _c , independently of one another, representing an alkyl, alkoxy, carbonyl, or oxycarbonyl group, linear or branched, these groups being as defined above, or an aromatic or aliphatic cycle, these cycles being as defined above, or
  • when R ₁ and R ₂ , or R ₃ and R ₄ , and / or R ₄ and R ₅ , and / or R ₇ and R ₈ , and / or R ₈ and R ₉ , and / or R ₉ and R ₁₀ , do not represent the different atoms or groups or rings mentioned above, then R ₁ in association with R ₂ , or R ₂ in association with R ₃ , and / or R ₃ in association with R ₄ , and / or R ₄ in association with R ₅ , and / or R ₇ in association with R ₈ , and / or R ₈ in association with R ₉ , and / or R ₉ in association with R ₁₀ , form respectively with C ₁ and C ₂ , or with C ₂ and C ₃ , or with C ₃ and C ₄ , or with C ₄ , C _4a and C ₅ , or with C ₇ and C ₈ , or with C ₈ and C ₉ , or with C ₉ and C ₁₀ , a cycle aromatic or aliphatic of 5 to 10 carbon atoms, this ring being optionally substituted, in particular by a halogen, and / or by an alkyl, alkoxy, carbonyl, or oxycarbonyl group, and / or by an aromatic or aliphatic ring, these groups or cycles being as defined above, or
  • when R ₃ and R ₄ do not represent the different atoms or groups or rings mentioned above, then R ₃ in association with R ₄ form an indole group of formula
    

in which R _a is as defined above,

• Y represents:
  • a group -OR _d , R _d representing a hydrogen atom, or an alkyl, carbonyl or oxycarbonyl group, linear or branched, these groups being as defined above, or an aromatic or aliphatic ring, these cycles being such as defined above, or
  • a group -NR _e R _f , R _e and R _f independently of one another, representing an alkyl, alkoxy, carbonyl, or oxycarbonyl group, linear or branched, these groups being as defined above, or a aromatic or aliphatic cycle, these cycles being as defined above,
  • it being understood that when R _d , or at least one of R _e or of R _f , do not represent one of the different atoms or groups or rings mentioned above, then R _d , or at least one of R _e or R _f , in association with R ₅ , or in association with R ₇ , form respectively with C ₅ and C ₆ , or with C ₆ , C _6a and C ₇ , an aromatic or aliphatic heterocycle of 5 to 10 atoms carbon, where appropriate substituted, in particular by a halogen, and / or by an alkyl, alkoxy, carbonyl, or oxycarbonyl group, and / or by an aromatic or aliphatic ring, these groups or rings being as defined above,
  • X represents an atom in anionic form, such as a halogen atom, in particular a bromine or chlorine atom, or a group of atoms in anionic form, such as a perchlorate, and the nitrogen of the heterocycle A of formula (I) is in quaternary form and is linked on the one hand by covalent bond to carbon in position 11, and, on the other hand, by ionic bond to X defined above,
  • it being understood that when R ₁ and R ₁₀ do not represent one of the different atoms or groups or rings mentioned above, then R ₁ in association with R ₁₀ form with C ₁ , the nitrogen of heterocycle A of the formula (I), C ₁₁ , and C ₁₀ , an aromatic or aliphatic heterocycle of 5 to 10 carbon atoms, optionally substituted, in particular by a halogen, and / or by an alkyl, alkoxy, carbonyl group; or oxycarbonyl, and / or by an aromatic or aliphatic cycle, these groups or cycles being as defined above, for the preparation of medicaments intended for the treatment of pathologies, in particular pulmonary, digestive or cardiac, linked to disorders of ionic flows transmembrane, in particular of chlorine and, where appropriate, of bicarbonate, in the organism (human or animal), in particular for the preparation of medicaments intended for the treatment of cystic fibrosis, or in the prevention of the rejection of cytotoxic drugs (in particular antitumor) , or the treatment of obstructions of the bronchial or digestive tracts (in particular pancreatic or intestinal)

   dans laquelle :

• R₁ et R₂ représentent un atome d'hydrogène, ou forment en association avec C₁ et C₂ un cycle aromatique à 6 atomes de carbone,
• Y représente un groupe -OH ou -NH₂, ou -NHCOCH₃,
• R₇, R₈, R₉ et R₁₀ représentent un atome d'hydrogène, ou l'un de R₇, R₈, R₉ ou R₁₀, représente un atome d'halogène, notamment un atome de chlore. de brome ou de fluor,
• X représente un atome d'halogène sous forme anionique, notamment un atome de brome Br^-, ou de chlore Cl^-, ou un groupe d'atomes sous forme anionique, notamment un perchlorate ClO₄ ^-.

The invention relates more particularly still to the use as described above, of compounds of general formula (IIIa) below:

in which :

• R ₁ and R ₂ represent a hydrogen atom, or form in association with C ₁ and C ₂ an aromatic ring with 6 carbon atoms,
• Y represents a group -OH or -NH ₂ , or -NHCOCH ₃ ,
• R ₇ , R ₈ , R ₉ and R ₁₀ represent a hydrogen atom, or one of R ₇ , R ₈ , R ₉ or R ₁₀ , represents a halogen atom, in particular a chlorine atom. bromine or fluorine,
• X represents a halogen atom in anionic form, in particular a bromine atom Br ^- , or chlorine Cl ^- , or a group of atoms in anionic form, in particular a perchlorate ClO ₄ ^- .

• R₁ et R₂ représentent un atome d'hydrogène,
• Y représente un groupe -OH,
• X représente un atome d'halogène sous forme anionique, notamment un atome de brome Br^-, ou de chlore Cl^-, ou un groupe d'atomes sous forme anionique, notamment un perchlorate ClO₄ ^-,
• R₇, R₈, R₉ et R₁₀ représentent indépendamment les uns des autres un atome d'hydrogène ou un atome d'halogène, notamment un atome de chlore, de brome ou de fluor.

Particularly preferred compounds in the context of the present invention are those of formula (IIIa) in which:

• R ₁ and R ₂ represent a hydrogen atom,
• Y represents a group -OH,
• X represents a halogen atom in anionic form, in particular a bromine atom Br ^- , or chlorine Cl ^- , or a group of atoms in anionic form, in particular a perchlorate ClO ₄ ^- ,
• R ₇ , R ₈ , R ₉ and R ₁₀ independently of one another represent a hydrogen atom or a halogen atom, in particular a chlorine, bromine or fluorine atom.

Compounds of general formula (IIIa) advantageously used in the context of the present invention, are those chosen from the following:

   dans laquelle R_a, R₁, R₂, R₅, R₇, R₈, R₉, R₁₀, X et Y sont tels que définis ci-dessus, et notamment les composés de formule (IIIb) dans laquelle :

• R_a représente un atome d'hydrogène,
• R₁ et R₂ représentent un atome d'hydrogène, et il n'y a pas de double liaison entre les deux carbones portant R₁ et R₂,
• R₅ représente un atome d'hydrogène,
• R₇, R₈, R₉ et R₁₀ représentent un atome d'hydrogène, ou l'un de R₇, R₈, R₉ ou R₁₀ représente un atome d'halogène, notamment un atome de chlore, de brome ou de fluor,
• Y représente -NH₂,
• X représente un atome d'halogène sous forme anionique, notamment un atome de brome Br^-, ou de chlore Cl^-, ou un groupe d'atomes sous forme anionique, notamment un perchlorate ClO₄ ^-.

The invention relates more particularly still to the use, as described above, of compounds of general formula (IIIb) below:

in which R _a , R ₁ , R ₂ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₀ , X and Y are as defined above, and in particular the compounds of formula (IIIb) in which:

• R _a represents a hydrogen atom,
• R ₁ and R ₂ represent a hydrogen atom, and there is no double bond between the two carbons carrying R ₁ and R ₂ ,
• R ₅ represents a hydrogen atom,
• R ₇ , R ₈ , R ₉ and R ₁₀ represent a hydrogen atom, or one of R ₇ , R ₈ , R ₉ or R ₁₀ represents a halogen atom, in particular a chlorine, bromine atom or fluorine,
• Y represents -NH ₂ ,
• X represents a halogen atom in anionic form, in particular a bromine atom Br ^- , or chlorine Cl ^- , or a group of atoms in anionic form, in particular a perchlorate ClO ₄ ^- .

• composé G : R₇ = Cl, R₈ = R₉ = R₁₀ = H,
• composé H : R₇ = R₈ = R₉ = R₁₀ = H,
• composé 1 : R₈ = Cl, R₇ = R₉ = R₁₀ = H,
• composé J : R₉ = Cl, R7 = R₈ = R₁₀ = H,
• composé K : R₁₀ = Cl, R₇ = R₈ = R₉ = H,
• composé L : R₉ = Br, R₇ = R₈ = R₁₀ = H.

Compounds of formula (IIIb) advantageously used in the context of the present invention are those chosen from the following:

• compound G: R ₇ = Cl, R ₈ = R ₉ = R ₁₀ = H,
• compound H: R ₇ = R ₈ = R ₉ = R ₁₀ = H,
• compound 1: R ₈ = Cl, R ₇ = R ₉ = R ₁₀ = H,
• compound J: R ₉ = Cl, R7 = R ₈ = R ₁₀ = H,
• compound K: R ₁₀ = Cl, R ₇ = R ₈ = R ₉ = H,
• compound L: R ₉ = Br, R ₇ = R ₈ = R ₁₀ = H.

The invention also relates to any pharmaceutical composition comprising, as active principle (s), at least one of the compounds of formula general (III) described above, in association with a physiologically vehicle acceptable.

The subject of the invention is more particularly any composition pharmaceutical as described above, comprising, as a principle (s) active (s), at least one of the compounds of formula (III), and more particularly of formula (IIIa), as defined above, and in particular at least one of compounds 11 to 27 described above, and more particularly still of formula (IIIb), as defined above, and in particular at least one of the compounds G to L described above.

Preferred pharmaceutical compositions of the invention are those comprising compound 19 (also designated MPB-07), where appropriate in association with one (or more) other compound (s) of the invention described above.

Advantageously, the pharmaceutical compositions according to the invention are present in a form which can be administered orally, in particular in the form tablets, or capsules, or in an administrable form parenteral, especially in the form of injectable preparations intravenous, intramuscular, or subcutaneous, or even by air, especially by pulmonary route in the form of aerosols.

Advantageously also, the pharmaceutical compositions according to the invention are characterized in that the amounts of active ingredient (s) are such that the daily dosage in active ingredient (s) is approximately 0.1 mg / kg at 5 mg / kg, in particular approximately 3 mg / kg, in one or more doses.

The invention also relates to the compounds of general formula (III) described above, as such, with the exclusion of the compounds of the following formulas:

The invention relates more particularly to the compounds of formula general (IIIa) described above, including in particular compounds 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 26 and 27 described above.

The invention relates more particularly still to the compounds of general formula (IIIb) described above, including in particular compounds G to L described above.

• traitement du dérivé de formule (A) dans laquelle R₁, R₂, R₃, R₄; et R₅, sont tels que définis dans la formule (III), par le phényllithium ou le diisopropyl amidure de lithium, avantageusement dans l'éther ou le THF, ce qui conduit à l'obtention de dérivés de formule (B) dans laquelle R₁, R₂, R₃, R₄, et R₅, sont tels que définis dans la formule (III), selon le schéma réactionnel suivant:
  
• condensation du dérivé de formule (B) obtenu à l'étape précédente avec le dérivé de formule (C) dans laquelle R₇, R₈, R₉, R₁₀, et X sont tels que définis dans la formule (III), ce qui conduit à l'obtention de dérivés de formule (B) dans laquelle R₁, R₂, R₃, R₄, R₅, R₇, R₈, R₉, R₁₀, et X sont tels que définis dans la formule (III), selon le schéma réactionnel suivant :
  
• traitement du composé de formule (D) par addition d'H₂O, ce qui conduit à l'obtention du dérivé de formule (II) suivante, dans laquelle R₁ à R₅, R₇ à R₁₀, et X sont tels que définis dans la formule (III), et Y représente -NH₂,
  
• le cas échéant, traitement du composé de formule (II) susmentionnée, par un dérivé comportant les groupes R_e et R_f tels que définis dans la formule (III) ce dérivé étant susceptible de réagir avec l'atome d'azote lié au carbone en position 6 du composé de formule (II) susmentionnée, notamment par un halogénure de R_e et/ou de R_f tout en ayant, si nécessaire, pris soin de protéger au préalable celles des autres fonctions présentes sur le composé de formule (II) susmentionnée et susceptibles de réagir avec le dérivé comportant les groupes R_e et R_f susmentionnés, ce qui conduit à l'obtention du composé de formule (II) suivante dans laquelle R₁ à R₅, R₇ à R₁₀, et X sont tels que définis ci-dessus, et Y représente un groupe -NR_eR_f tel que défini dans la formule (III)
  
• le cas échéant, hydrolyse, notamment par action de l'acide sulfurique (pH3) à 40°C, du composé de formule (II) susmentionnée dans laquelle Y représente NH₂, ce qui conduit à l'obtention du composé de formule (II) suivante, correspondant à un dérivé de formule (II) décrite ci-dessus, dans laquelle R₁ à R₅, R₇ à R₁₀, et X sont tels que définis dans la formule (III) et Y représente un groupe -OH,
  
• le cas échéant, traitement du composé de formule (II) susmentionnée, dans laquelle Y représente un groupe -OH, par un dérivé comportant le groupe R_d, tel que défini dans la formule (III), ce dérivé étant susceptible de réagir avec l'atome d'oxygène lié au carbone en position 7 du composé de formule (II) susmentionnée, notamment par un halogénure de R_d, tout en ayant, si nécessaire, pris soin de protéger au préalable celles des autres fonctions présentes sur le composé de formule (II) susmentionnée et susceptibles de réagir avec le dérivé comportant le groupe R_d susmentionné, ce qui conduit à l'obtention du composé de formule (II) suivante dans laquelle R₁ à R₅, R₇ à R₁₀, et X sont tels que définis ci-dessus, et Y représente un groupe -OR_d tel que défini dans la formule (III),
  
• le cas échéant, chauffage, avantageusement à 200°C, des composés de formule (II) susmentionnée dans laquelle Y représente -NH₂ ou -OH, ce qui conduit respectivement aux composés de formules (III) suivantes, correspondant aux composés de formule (III) décrite ci-dessus, dans laquelle R₁ à R₅, R₇ à R₁₀, et X sont tels que définis dans la formule (III), et Y représente un groupe -NH₂ ou -OH,
  
  
• le cas échéant, traitement du composé de formule (III) susmentionnée, dans laquelle Y représente un groupe -NH₂, par un dérivé comportant les groupes R_e et R_f tels que définis dans la formule (III), ce dérivé étant susceptible de réagir avec l'atome d'azote lié au carbone en position 6 du composé de formule (III) susmentionnée, notamment par un halogénure de R_e et/ou de R_f, tout en ayant, si nécessaire, pris soin de protéger au préalable celles des autres fonctions présentes sur le composé de formule (III) susmentionnée et susceptibles de réagir avec le dérivé comportant les groupes R_e et R_f susmentionnés, ce qui conduit à l'obtention du composé de formule (III) suivante dans laquelle R₁ à R₅, R₇ à R₁₀, et X sont tels que définis ci-dessus, et Y représente un groupe -NR_eR_f tel que défini dans la formule (III),
  
• le cas échéant, traitement du composé de formule (III) susmentionnée, dans laquelle Y représente un groupe -OH, par un dérivé comportant le groupe R_d, tel que défini dans la formule (III), ce dérivé étant susceptible de réagir avec l'atome d'oxygène lié au carbone en position 7 du composé de formule (III) susmentionnée, notamment par un halogénure de R_d, tout en ayant, si nécessaire, pris soin de protéger au préalable celles des autres fonctions présentes sur le composé de formule (III) susmentionnée et susceptibles de réagir avec le dérivé comportant le groupe R_d susmentionné, ce qui conduit à l'obtention du composé de formule (III) suivante dans laquelle R₁ à R₅, R₇ à R₁₀, et X sont tels que définis ci-dessus, et Y représente un groupe -OR_d tel que défini dans la formule (III),
  
• le cas échéant, hydrogénation des composés de formules (II) ou (III) susmentionnées, notamment par hydrogénation catalytique en présence d'oxyde de platine à pression réduite, ce qui conduit à l'obtention des composés de formules (II) ou (III) suivantes :
  
  

   dans lesquelles R₁ à R₅, R₇ à R₁₀, X et Y sont tels que définis ci-dessus.The subject of the invention is also a process for the preparation of the compounds of general formula (III), characterized in that it comprises the following stages:

• treatment of the derivative of formula (A) in which R ₁ , R ₂ , R ₃ , R ₄ ; and R ₅ , are as defined in formula (III), by phenyllithium or lithium diisopropyl amide, advantageously in ether or THF, which leads to obtaining derivatives of formula (B) in which R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ , are as defined in formula (III), according to the following reaction scheme:
  
• condensation of the derivative of formula (B) obtained in the previous step with the derivative of formula (C) in which R ₇ , R ₈ , R ₉ , R ₁₀ , and X are as defined in formula (III), this which leads to obtaining derivatives of formula (B) in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₀ , and X are as defined in formula (III), according to the following reaction scheme:
  
• treatment of the compound of formula (D) by addition of H ₂ O, which leads to obtaining the derivative of formula (II) below, in which R ₁ to R ₅ , R ₇ to R ₁₀ , and X are such as defined in formula (III), and Y represents -NH ₂ ,
  
• where appropriate, treatment of the above-mentioned compound of formula (II) with a derivative comprising the groups R _e and R _f as defined in formula (III), this derivative being capable of reacting with the nitrogen atom bonded to carbon in position 6 of the compound of formula (II) above, in particular by a halide of R _e and / or of R _f while having, if necessary, taken care to protect beforehand those of the other functions present on the compound of formula (II ) mentioned above and capable of reacting with the derivative comprising the above mentioned groups R _e and R _f , which leads to obtaining the following compound of formula (II) in which R ₁ to R ₅ , R ₇ to R ₁₀ , and X are as defined above, and Y represents a group -NR _e R _f as defined in formula (III)
  
• where appropriate, hydrolysis, in particular by the action of sulfuric acid (pH3) at 40 ° C, of the above-mentioned compound of formula (II) in which Y represents NH ₂ , which leads to obtaining the compound of formula (II ) following, corresponding to a derivative of formula (II) described above, in which R ₁ to R ₅ , R ₇ to R ₁₀ , and X are as defined in formula (III) and Y represents a group -OH ,
  
• where appropriate, treatment of the above-mentioned compound of formula (II), in which Y represents an —OH group, with a derivative comprising the group R _d , as defined in formula (III), this derivative being capable of reacting with l oxygen atom bonded to the carbon in position 7 of the compound of formula (II) above, in particular by a halide of R _d , while having, if necessary, taken care to protect beforehand those of the other functions present on the compound of aforementioned formula (II) and capable of reacting with the derivative comprising the aforementioned group R _d , which leads to the production of the following compound of formula (II) in which R ₁ to R ₅ , R ₇ to R ₁₀ , and X are as defined above, and Y represents a group -OR _d as defined in formula (III),
  
• where appropriate, heating, advantageously at 200 ° C., of the compounds of the above-mentioned formula (II) in which Y represents -NH ₂ or -OH, which leads respectively to the compounds of the following formulas (III), corresponding to the compounds of formula ( III) described above, in which R ₁ to R ₅ , R ₇ to R ₁₀ , and X are as defined in formula (III), and Y represents a group -NH ₂ or -OH,
  
  
• where appropriate, treatment of the above-mentioned compound of formula (III), in which Y represents an —NH ₂ group, with a derivative comprising the groups R _e and R _f as defined in formula (III), this derivative being capable of react with the nitrogen atom bonded to the carbon in position 6 of the compound of formula above-mentioned, in particular with a halide of R _e and / or R _f , while having, if necessary, taken care to protect beforehand those of the other functions present on the above-mentioned compound of formula (III) and capable of reacting with the derivative comprising the above-mentioned groups R _e and R _f , which leads to the production of the following compound of formula (III) in which R ₁ to R ₅ , R ₇ to R ₁₀ , and X are as defined above, and Y represents a group -NR _e R _f as defined in formula (III),
  
• where appropriate, treatment of the above-mentioned compound of formula (III), in which Y represents an —OH group, with a derivative comprising the group R _d , as defined in formula (III), this derivative being capable of reacting with l oxygen atom bonded to the carbon in position 7 of the compound of formula aforementioned, in particular by a halide of R _d , while having, if necessary, taken care to protect beforehand those of the other functions present on the compound of above-mentioned formula (III) and capable of reacting with the derivative comprising the above-mentioned group R _d , which leads to the production of the following compound of formula (III) in which R ₁ to R ₅ , R ₇ to R ₁₀ , and X are as defined above, and Y represents a group -OR _d as defined in formula (III),
  
• where appropriate, hydrogenation of the compounds of formulas (II) or (III) above, in particular by catalytic hydrogenation in the presence of platinum oxide at reduced pressure, which leads to obtaining the compounds of formulas (II) or (III ) following:
  
  

in which R ₁ to R ₅ , R ₇ to R ₁₀ , X and Y are as defined above.

The compounds A to L described above are advantageously obtained by treatment of the harmalane with butyl lithium (Buli, 2 eq) at -40 ° C, then addition of 2-chlorobenzonitrile (optionally substituted with one or more of groups R ₇ , R ₈ , R ₉ and R ₁₀ as defined above), which leads to obtaining the compounds of formula A to F which, by heating to 195 ° C. under nitrogen, lead respectively to compounds G to L.

The invention will be further illustrated by means of the detailed description which follows processes for the preparation of compounds 1 to 24 described above, as well as studying the effects of some of these compounds on CFTR.

I- Processes for the preparation of compounds 1 to 24: a) 1-hydroxy, 1-phenyl, 2- (2-pyridyl) ethylene (compound 1)

Compound 1 is prepared according to a procedure identical to that described below in the context of the preparation of compound 2, but with the use of benzonitrile instead of 2-chlorobenzonitrile.

b) 1-hydroxy, 1- (2-chlorophenyl), 2- (2-pyridyl) ethylene (compound 2)

• Point de fusion (Pf) = 190 °C
• Analyse élémentaire : C₁₃ H₁₁ Cl N₂ O Calculé % C : 58,20 H : 4,10 N : 5,20 Trouvé % C : 58,00 H : 4,10 N : 5,30

2.22 g (0.022 mol) of diisopropylamine in 30 ml of anhydrous THF are placed in a 500 ml reactor, fitted with a reflux condenser with a calcium chloride guard, and a nitrogen inlet. The solution is brought to 0 ° C. and then 13.75 ml of BuLi is added in solution at 1.6 M in hexane (0.022 mole). The mixture is stirred for 30 minutes at 0 ° C. and then the temperature is lowered to -40 ° C., then 1.86 g (0.02 mole) of 2-methylpyridrine are added. The mixture is stirred for 30 minutes at -40 ° C., then 2.75 g of 2-chlorobenzonitrile is added in 20 ml of anhydrous THF. The reaction medium is allowed to return to ambient temperature and 20 ml of water are added, then the pH is adjusted to around 2 by addition of 2N H ₂ SO ₄ . The mixture is heated to reflux and with stirring for 1 hour. Extracted with chloroform, dried over sodium sulfate. The solvent is evaporated off and the residue is dissolved in a minimum of anhydrous ether, then ethanol saturated with HCl is added dropwise until complete precipitation of the hydrochloride. 2.99 g of compound 2 are thus obtained, ie a yield of 56%

• Melting point (Pf) = 190 ° C
• Elementary analysis: C ₁₃ H ₁₁ Cl N ₂ O Calculated% C: 58.20 H: 4.10 N: 5.20 Find % C: 58.00 H: 4.10 N: 5.30

c) 1-amino, 1- (2-chlorophenyl), 2- (2-pyridyl) ethylene (compound 3)

Compound 3 is prepared according to a procedure identical to that described above in the context of the preparation of compound 2, but without the hydrolysis step by addition of H ₂ SO ₄ .

d) 1-hydroxy, 1- (2-bromophenyl) 2- (2-pyridyl) ethylene (compound 4)

Compound 4 is prepared according to a procedure identical to that described above in the context of the preparation of compound 2, but with the use of 2-bromobenzonitrile instead of 2-chlorobenzonitrile.

e) 1-hydroxy 1- (2,3-dichlorophenyl), 2- (2-pyridyl) ethylene (compound 5)

• Point de fusion (Pf) = 112°C
• Analyse élémentaire : C₁₃ H₉ N O Cl₂ Calculé % C : 58,67 H : 3,41 N : 5,26 Trouvé % C : 58,53 H : 3,63 N : 5,34
• Spectre de ¹H RMN (CDCl₃) (δ ppm, signal, N protons, attribution) : 8, doublet, H en 6 pyridine ; 7,6,5, multiplet, 6, H aromatiques. 5,55, s, 1 ( 80%) H vinylique ; 4,25, s s2 (20 %) CH₂.

2.22 g (0.022 mol) of diisopropylamine in 30 ml of anhydrous THF are placed in a 500 ml reactor, fitted with a reflux condenser with a calcium chloride guard, and a nitrogen inlet. The solution is brought to 0 ° C. and then 13.75 ml of BuLi is added in solution at 1.6 M in hexane (0.022 mole). The mixture is stirred for 30 minutes at 0 ° C., then the temperature is lowered to -40 ° C., then 1.86 g (0.02 mole) of 2-methylpyridrine are added. The mixture is stirred for 30 minutes at -40 ° C., then 2.58 g (0.015 mol) of 2,3-dichlorobenzonitrile in 20 ml of anhydrous THF are added. The reaction medium is allowed to return to room temperature and 20 ml of water are added, then the pH is adjusted to around 2 by addition of 2N H ₂ SO ₄ . The mixture is heated to reflux and with stirring for 2 hours. The organic phase is separated and dried over sodium sulfate. The solvent is evaporated off and the residue is chromatographed on a silica column, eluting with chloroform to obtain 3.19 g (80%) of pure ketone.

• Melting point (mp) = 112 ° C
• Elementary analysis: C ₁₃ H ₉ NO Cl ₂ Calculated% C: 58.67 H: 3.41 N: 5.26 Find % C: 58.53 H: 3.63 N: 5.34
• ¹ H NMR spectrum (CDCl ₃ ) (δ ppm, signal, N protons, attribution): 8, doublet, H in 6 pyridine; 7,6,5, multiplet, 6, H aromatics. 5.55, s, 1 (80%) vinyl H; 4.25, s s2 (20%) CH ₂ .

• Analyse élémentaire : C₁₃ H₁₀ N O Cl₃ : Calculé % C : 51,60 H : 3,33 N : 4,63 Trouvé % C : 51,75 H : 3,52 N : 4,58

The base thus obtained can be transformed into the hydrochloride: the base is dissolved in anhydrous ether and anhydrous ethanol saturated with HCl is added dropwise.

• Elementary analysis: C ₁₃ H ₁₀ NO Cl ₃ : Calculated% C: 51.60 H: 3.33 N: 4.63 Find % C: 51.75 H: 3.52 N: 4.58

f) 1-hydroxy, 1- (2,4-dichlorophenyl), 2- (2-pyridyl) ethylene (compound 6)

Compound 6 is prepared according to a procedure identical to that described above in the context of the preparation of compound 2, but with the use of 2-4-dichiorobenzonitrile instead of 2-chlorobenzonitrile.

g) 1-hydroxy, 1- (2,5-dichlorophenyl), 2- (2-pyridyl) ethylene (compound 7)

Compound 7 is prepared according to a procedure identical to that described above in the context of the preparation of compound 2, but with the use of 2-5-dichlorobenzonitrile instead of 2-chlorobenzonitrile.

h) 1-hydroxy, 1- (2,6-dichlorophenyl), 2- (2-pyridyl) ethylene (compound 8)

Compound 8 is prepared according to a procedure identical to that described above in the context of the preparation of compound 2, but with the use of 2-6-dichlorobenzonitrile instead of 2-chlorobenzonitrile.

i) 1-hydroxy, 1- (2-chlorophenyl), 2- (2-quinonyl) ethylene (compound 9)

Compound 9 is prepared according to a procedure identical to that described above in the context of the preparation of compound 2, but with the use of 2-methylquinoline instead of 2-methylpyridine.

j) 1-amino, 1- (2-chlorophenyl), 2- (2-quinolyl) ethylene (compound 10)

Compound 10 is prepared according to a procedure identical to that described above in the context of the preparation of compound 9, but without the hydrolysis step by addition of H ₂ SO ₄ .

k) 6-aminobenzo [c] quinolizium chloride MPB-26 (compound 11)

• Point de fusion (Pf) = décomposition vers 280°C
• Analyse élémentaire : C₁₃ H₁₃ C₁ N₂ O (C₁₃ H₁₁ C₁ N₂, H₂O) Calculé % C : 62,78 H : 5,27 N: 11,26 Trouvé % C : 62,86 H : 5,03 N : 11,25
• Spectre de masse : m/e 194 (M⁺ - HCl - H₂O).
• Spectre infrarouge (KBr) (ν cm^-1, attribution) : 3460, 3360, NH₂; 1660, 1640, 1600, C=N, C=C ; 760 benzène orthosubstitué.
• Spectre de ¹H RMN (DMSOd6) (δ ppm, signal, n protons, attribution): 3,2 à 4, pic échangeable D₂O (NH₂ + H₂O); 7,1 singulet, H en 5; 7,4 à 8,15, 2 massifs, 5H aromatiques; 9,00, 2H, aromatiques ; 9,8, doublet J=8 Hz, H en 1.

2.22 g (0.022 mol) of diisopropylamine in 30 ml of anhydrous THF are placed in a 500 ml reactor, fitted with a reflux condenser with a calcium chloride guard, and a nitrogen inlet. The solution is brought to 0 ° C., then 13.75 ml of BuLi is added in solution at 1.6 M in hexane (0.022 mole). The mixture is stirred for 30 minutes at 0 ° C., then the temperature is lowered to -40 ° C., then 1.86 g (0.02 mole) of 2-methylpyridrine are added. The mixture is stirred for 30 minutes at -40 ° C., then 2.75 g of 2-chlorobenzonitrile (0.02 mole) are added in 20 ml of anhydrous THF. The reaction medium is allowed to return to ambient temperature and a 10% solution of ammonium chloride is added. The organic phase is separated, dried over SO ₄ Na ₂ , evaporated and the residue is brought to 200 ° C. under a stream of nitrogen for 15 minutes. A release of white vapors is observed and the residue takes on a brownish mass. The product is purified by a first wash with acetone then dissolution in ethanol and precipitated with ethyl acetate. It can also be purified by chromatography on a neutral alumina column and elution with ethyl acetate. This gives 1.73 g (35%) of a product which crystallizes with one molecule of water.

• Melting point (Pf) = decomposition around 280 ° C
• Elementary analysis: C ₁₃ H ₁₃ C ₁ N ₂ O (C ₁₃ H ₁₁ C ₁ N ₂ , H ₂ O) Calculated% C: 62.78 H: 5.27 N: 11.26 Find % C: 62.86 H: 5.03 N: 11.25
• Mass spectrum: m / e 194 (M ⁺ - HCl - H ₂ O).
• Infrared spectrum (KBr) (ν cm ^-1 , attribution): 3460, 3360, NH ₂ ; 1660, 1640, 1600, C = N, C = C; 760 orthosubstituted benzene.
• ¹ H NMR spectrum (DMSOd6) (δ ppm, signal, n protons, attribution): 3.2 to 4, exchangeable peak D ₂ O (NH ₂ + H ₂ O); 7.1 singlet, H in 5; 7.4 to 8.15, 2 massive, 5H aromatic; 9.00, 2H, aromatics; 9.8, doublet J = 8 Hz, H in 1.

l) 6-Hydroxybenzo [c] quinolizinium chloride MPB-05 (compound 12)

• Point de fusion (Pf) = 256°C (décomposition)
• Analyse élémentaire : C₁₃ H₁₀ Cl N O, H₂O soit C₁₃ H₁₂ Cl N O (M=231,5 + 18=249,5) Calculé % C : 62,53 H : 4,85 N : 5,61 Trouvé % C : 62,40 H : 5,00 N : 5,80
• Spectre de masse : m/e 195 (M⁺ - HCl - H₂O), 167 (M⁺ - HCl - H₂O - CO).
• Spectre infrarouge (KBr) (ν cm^-1, attribution) : 3250, OH; 1640, C=N, 770, benzène orthosubstitué.
• Spectre de ¹H RMN (DMSOd6) (δ ppm, signal, n protons, attribution): 6,60, pic large échangeable D₂O, HO; 7,75, singulet, H en 5 ; 7,9 à 8,6, multiplet, 6H, aromatiques; 9,15, doublet, J = 6,5 Hz, 1H ; 10, doublet, J = 6Hz, H en 1.

1-hydroxy, 1- (2-chlorophenyl), 2- (2-pyridyl) ethylene (compound 2) is neutralized with an aqueous solution of sodium carbonate, the base is extracted with ether, the solution is dried over SO ₄ Na ₂ then the solvent evaporated. 1.16 g (0.005 mole) of base in the form of a pale yellow oil is heated under nitrogen at 195 ° C for 15 minutes. The residue thus obtained is washed with acetone, then dissolved in ethanol and precipitated by addition of ethyl acetate. 0.75 g (60%) of a product crystallized with a molecule of creamy white water is obtained.

• Melting point (mp) = 256 ° C (decomposition)
• Elementary analysis: C ₁₃ H ₁₀ Cl NO, H ₂ O or C ₁₃ H ₁₂ Cl NO (M = 231.5 + 18 = 249.5) Calculated% C: 62.53 H: 4.85 N: 5.61 Find % C: 62.40 H: 5.00 N: 5.80
• Mass spectrum: m / e 195 (M ⁺ - HCl - H ₂ O), 167 (M ⁺ - HCl - H ₂ O - CO).
• Infrared spectrum (KBr) (ν cm ^-1 , attribution): 3250, OH; 1640, C = N, 770, orthosubstituted benzene.
• ¹ H NMR spectrum (DMSOd6) (δ ppm, signal, n protons, attribution): 6.60, broad exchangeable peak D ₂ O, HO; 7.75, singlet, H in 5; 7.9 to 8.6, multiplet, 6H, aromatic; 9.15, doublet, J = 6.5 Hz, 1H; 10, doublet, J = 6Hz, H in 1.

m) 6-Aminobenzo [c] quinolizinium bromide MPB-01 (compound 13):

Compound 13 is prepared according to a procedure identical to that described above in the context of the preparation of compound 11, but with use of 2-bromobenzonitrile instead of 2-chlorobenzonitrile.

n) 6-amino, 10-chlorobenzo [c] quinolizinium chloride MPB-02 (compound 14):

Compound 14 is prepared according to a procedure identical to that described above in the context of the preparation of compound 11, but with use of 2,3-dichlorobenzonitrile instead of 2-chlorobenzonitrile.

o) 6-amino, 9-chlorobenzo [c] quinolizinium chloride (compound 15):

Compound 15 is prepared according to a procedure identical to that described above in the context of the preparation of compound 11, but with use of 2,4-dichlorobenzonitrile instead of 2-chlorobenzonitrile.

p) 6-amino, 7-chlorobenzo [c] quinolizinium chloride (compound 16):

Compound 16 is prepared according to a procedure identical to that described above in the context of the preparation of compound 11, but with use of 2,6-dichlorobenzonitrile instead of 2-chlorobenzonitrile.

q) 6-amino, 8-chlorobenzo [c] quinolizinium chloride (compound 17):

Compound 17 is prepared according to a procedure identical to that described above in the context of the preparation of compound 11, but with use of 2,5-dichlorobenzonitrile instead of 2-chlorobenzonitrile.

r) 6-Hydroxybenzo [c] quinolizinium bromide MPB-06 (compound 18):

Compound 18 is prepared according to a procedure identical to that described below in the context of the preparation of compound 19, but carried out from compound 4 instead of compound 5.

s) 6-hydroxy, 10-chlorobenzo [c] quinolizinium chloride MPB-07 (compound 19):

• Pf = 196 °C (décomposition)
• Analyse élémentaire : C₁₃ H₁₀ N O Cl₂ Calculé % C : 56,75 H : 3,66 N : 4,63 Trouvé % C : 56,25 H : 3,31 N : 4,78

1-hydroxy, 1- (2-bromophenyl), 2- (2-pyridyl) ethylene (compound 5) 1.40 g (0.0053 mole) is heated under nitrogen to 215 ° C. Around 190 ° C, the appearance of white HCI fumes is noted and the heating is continued for 10 minutes at 220 ° C. The product is washed with chloroform and the residue (1.82 g) is purified by chromatography on a silica column, eluting with acetate and alcohol. 0.58 g (42%) of product is thus obtained.

• Mp = 196 ° C (decomposition)
• Elementary analysis: C ₁₃ H ₁₀ NO Cl ₂ Calculated% C: 56.75 H: 3.66 N: 4.63 Find % C: 56.25 H: 3.31 N: 4.78

t) 6-hydroxy, 9-chlorobenzo [c] quinolizinium chloride MPB-08 (compound 20):

Compound 20 is prepared according to a procedure identical to that described above in the context of the preparation of compound 19, but carried out at from compound 6 instead of compound 5.

u) 6-hydroxy, 7-chlorobenzo [c] quinolizium chloride (compound 21):

Compound 21 is prepared according to a procedure identical to that described above in the context of the preparation of compound 19, but carried out at from compound 8 instead of compound 5.

v) 8-Aminodibenzo [c, f] quinolizinium perchlorate (compound 22):

• Analyse élémentaire : C₁₇ H₁₃ Cl N₂ O₄ Calculé % C : 59,22 H : 3,80 N : 8,13 Trouvé % C : 59,39 H : 3,95 N : 8,26
• Spectre infrarouge (Kbr) (ν cm-¹, attribution) : 3400, 3280, NH₂; 1650, 1600, 1000, bande large, ClO₄.
• Spectre de ¹H RMN (DMSOd6) (δ ppm, signal, n protons, attribution) : 7,0, singulet, 1H en 5 ; 7,5 à 8,7, multiplet, 10H aromatiques ; 9,0 singulet, 2H, échangeables D₂O.

2.22 g (0.022 mol) of diisopropylamine in 30 ml of anhydrous THF are placed in a 500 ml reactor, fitted with a reflux condenser with a calcium chloride guard, and a nitrogen inlet. The solution is brought to 0 ° C., then 13.75 ml of BuLi is added in solution at 1.6 M in hexane (0.022 mole). The mixture is stirred for 30 minutes at 0 ° C., then the temperature is lowered to -40 ° C., then 2.86 g (0.02 mol) of quinaldin are added. The mixture is stirred for 30 minutes at -40 ° C., then 2.75 g of 2-chlorobenzonitrile (0.02 mole) are added in 20 ml of anhydrous THF. The reaction medium is allowed to return to ambient temperature and a 10% solution of ammonium chloride is added. The organic phase is separated, dried over SO ₄ Na ₂ , evaporated and the residue is brought to 230 ° C. under a stream of nitrogen for 30 minutes. A white vapor of hydrochloric acid is observed and the residue takes a brownish mass. The product is purified by a first wash with acetone then dissolution in ethanol and precipitated with ethyl acetate. The product is dissolved in a minimum of water and a solution of perchloric acid is added until the end of precipitation. The compound is filtered to collect 2.20 g (32%).

• Elementary analysis: C ₁₇ H ₁₃ Cl N ₂ O ₄ Calculated% C: 59.22 H: 3.80 N: 8.13 Find % C: 59.39 H: 3.95 N: 8.26
• Infrared spectrum (Kbr) (ν cm- ¹ , attribution): 3400, 3280, NH ₂ ; 1650, 1600, 1000, wide band, ClO ₄ .
• ¹ H NMR spectrum (DMSOd6) (δ ppm, signal, n protons, attribution): 7.0, singlet, 1H in 5; 7.5 to 8.7, multiplet, 10H aromatic; 9.0 singlet, 2H, exchangeable D ₂ O.

w) 6-acetamidobenzo [c] quinolizinium chloride (compound 23):

1 g (0.004 mole) of 6-aminobenzo [c] quinolizinium hydrochloride is dissolved in 10 ml of acetic acid, then 25 ml are added acetic anhydride. It is refluxed for 24 hours, then evaporated anhydride and acetic acid under reduced pressure.

• Pf = > à 280°C
• Analyse élémentaire : C₁₅ H₁₃ Cl N₂ O Calculé % C : 66,05 H : 4,80 N : 10,27 Trouvé % C : 65,85 H : 5,01 N : 10,31
• Spectre infrarouge (KBr) (ν cm^-1, attribution) : 3450, NH; 1700, C = O.

The purple-colored product obtained is washed with ethyl acetate and then recrystallized from ethanol. 0.93 g (85%) of a creamy white product is obtained.

• Mp => at 280 ° C
• Elementary analysis: C ₁₅ H ₁₃ Cl N ₂ O Calculated% C: 66.05 H: 4.80 N: 10.27 Find % C: 65.85 H: 5.01 N: 10.31
• Infrared spectrum (KBr) (ν cm ^-1 , attribution): 3450, NH; 1700, C = O.

x) 1,2,3,4-tetrahydro, 6-aminobenzo [c, f] quinolizinium perchlorate (compound 24):

• Pf = 240°C
• Analyse élémentaire : C₁₃ H₁₅ Cl N₂ O₄ Calculé % C : 52,27 H : 5,06 N : 9,38 Trouvé % C : 52,30 H : 5,16 N : 9,46
• Spectre infrarouge (KBr) (ν cm^-1, attribution) : 3460,3360,NH₂; 1650, 1600, 1100, bande large, CL0₄.
• Spectre de ¹H RMN (DMSOd6) (δ ppm, signal, n protons, attribution) : 2,1, multiplet, CH₂ en 2 et 3; 3,20, triplet, CH₂ en 4; 4,45, triplet, CH₂ en 1,; 6,60, singulet, H en 5; 7,6 à 8,4, multiplet, 4H, aromatiques; 8,6 singulet, 2H, échangeables D₂O.

6-aminobenzo [c] quinolizinium hydrochloride 0.50 g (0.002 mole) is dissolved in 20 ml of ethanol, and is placed in the presence of platinum oxide and hydrogen at atmospheric pressure. The hydrogenation is carried out in a few minutes then the solution is filtered, the alcohol evaporated and the residue taken up in 10 ml of distilled water and a 25% perchloric acid solution is added as long as the precipitate forms. The white product is recrystallized from methanol to give 0.54 g (91%) of perchlorate.

• Mp = 240 ° C
• Elementary analysis: C ₁₃ H ₁₅ Cl N ₂ O ₄ Calculated% C: 52.27 H: 5.06 N: 9.38 Find % C: 52.30 H: 5.16 N: 9.46
• Infrared spectrum (KBr) (ν cm ^-1 , attribution): 3460.3360, NH ₂ ; 1650, 1600, 1100, wide band, CL0 ₄ .
• ¹ H NMR spectrum (DMSOd6) (δ ppm, signal, n protons, attribution): 2.1, multiplet, CH ₂ in 2 and 3; 3.20, triplet, CH ₂ in 4; 4.45, triplet, CH ₂ in 1 ,; 6.60, singlet, H in 5; 7.6 to 8.4, multiplet, 4H, aromatic; 8.6 singlet, 2H, exchangeable D ₂ O.

II- Process for the preparation of compounds A to F: a) 1-amino 1- (2,6-dichlorophenyl) 2- [1- (3,4-dihydropyrido [3-4-b] indolyl)] ethylene (compound A)

• Pf = 228°C
• Analyse élémentaire : C₁₉ H₁₅ N₃ Cl₂ Calculé % C : 64,06 H : 4,24 N : 11,79 Trouvé % C : 64,21 H : 4,37 N : 11,62
• Spectre infrarouge (KBr) (ν cm^-1, attribution) :
• 3428, 3255, 3134 cm^-1, NH; NH₂
• 3134 cm^-1, C=C-H
• 2932, 2843 cm^-1, CH₂
• Spectre ¹ H RMN (CDCl₃) ( δ ppm, signal, n protons, attibution) :
• 8.40 singulet échangeable par D₂O, 3 H, NH et NH₂
• 7.20, multiplet, 7 H, protons aromatiques
• 5.00, singulet, 1 H, H vinylique
• 3.70, triplet, J=6Hz, 2 H, CH₂
• 3.00, triplet, J=6Hz, 2 H, CH₂

In a reactor fitted with a nitrogen inlet, 1.84 g (0.01 mole) of harmalane are dissolved in 40 ml of THF and are brought to -40 ° C. 13.75 ml (0.022 mole) of BuLi are added dropwise, a dark red coloration appears, the solution is left under stirring for 30 min. 2,6-dichlorobenzonitrile 1.71 g (0.01 mole) is dissolved in 15 ml of THF and then added dropwise. After 1 h at -40 ° C, the mixture is stirred for 4 h at laboratory temperature. The progress of the reaction is monitored on TLC, the disappearance of the starting materials is correlated with the appearance of a yellow fluorescent spot characteristic of imine. Hydrolysis with 5 ml of a 10% NH ₄ Cl solution makes it possible to collect the THF phase containing the imine. This phase is dried over NA ₂ SO ₄ , filtered and then evaporated to dryness. The product is purified by column chromatography on silica gel, elution with a CH ₂ Cl ₂ / CH ₃ COOC ₂ H ₅ 5% mixture. 2.06 g of compound A are thus obtained, ie a yield of 58%.

• Mp = 228 ° C
• Elementary analysis: C ₁₉ H ₁₅ N ₃ Cl ₂ Calculated% C: 64.06 H: 4.24 N: 11.79 Find % C: 64.21 H: 4.37 N: 11.62
• Infrared spectrum (KBr) (ν cm ^-1 , attribution):
• 3428, 3255, 3134 cm ^-1 , NH; NH ₂
• 3134 cm ^-1 , C = CH
• 2932, 2843 cm ^-1 , CH ₂
• ¹ H NMR spectrum (CDCl ₃ ) (δ ppm, signal, n protons, attibution):
• 8.40 singlet exchangeable by D ₂ O, 3 H, NH and NH ₂
• 7.20, multiplet, 7 H, aromatic protons
• 5.00, singlet, 1 H, H vinyl
• 3.70, triplet, J = 6Hz, 2H, CH ₂
• 3.00, triplet, J = 6Hz, 2H, CH ₂

b) 1-amino 1- (o-chlorophenyl) 2- [1- (3,4-dihydropyrido [3-4-b] indolyl)] ethylene (compound B)

Compound B is prepared according to a procedure identical to that described above in the context of the preparation of compound A but with the use of orthochlorobenzonitrile instead of 2.6 dichlorobenzonitrile.

c) 1-amino 1- (2,5-dichlorophenyl) 2- [1- (3,4-dihydropyrido [3-4-b] indolyl)] ethylene (compound C)

Compound C is prepared according to a procedure identical to that described above in the context of the preparation of compound A but with the use of 2.5 dichlorobenzonitrile instead of 2.6 dichlorobenzonitrile.

d) 1-amino 1- (2,4-dichiorophenyl) 2- [1- (3,4-dihydropyrido [3-4-b] indolyl)] ethylene (compound D)

Compound D is prepared according to a procedure identical to that described above in the context of the preparation of compound A but with the use of 2,4 dichlorobenzonitrile instead of 2,6 dichlorobenzonitrile.

e) 1-amino 1- (2,3-dichlorophenyl) 2- [1- (3,4-dihydropyrido [3-4-b] indolyl)) ethylene (compound E)

Compound E is prepared according to a procedure identical to that described above in the context of the preparation of compound A but with the use of 2,3 dichlorobenzonitrile instead of 2,6 dichlorobenzonitrile.

f) 1-amino 1- (4-bromo, 2-chlorophenyl) 2- [1- (3,4-dihydropyrido [3-4-b] indolyl)] ethylene (compound F)

Compound F is prepared according to a procedure identical to that described above in the context of the preparation of compound A but with the use of 2-chloro-4 bromobenzonitrile instead of 2,6 dichlorobenzonitrile.

III- Process for manufacturing compounds G to L: Cyclization of compounds A to F into quinolizinium G to L. a) 14-amino 6,7-dihydro chloride 12H-1-chloro benzo- [n] indolo [2,3-a] quinolizinium (compound G).

The purified enamine A is heated under nitrogen; around 150 ° C the product liquefies, then at 195 ° C white fumes appear and the product solidifies. The heating is maintained at this temperature for 10 min. The CCM shows the disappearance of the yellow fluorescent spot of the imine of an Rf of 0.3 on silica in CH ₂ Cl ₂ and the appearance of a yellow-green fluorescent spot of the cyclized product of an Rf of 0.1 on alumina in alcohol. The product is purified by washing with acetone, then recrystallization from alcohol or by chromatography: alumina - alcohol.

• Pf = 228°C.
• Analyse élémentaire : C₁₉ H₁₅ N₃ Cl₂, 1/2 H₂O Calculé % C : 62,48 H : 4,41 N : 11,50 Trouvé % C : 62,29 H : 4,47 N : 11,43
• Spectre ¹ H RMN (CF₃ COOD) ( δ ppm, signal, n protons, attibution) :
• 8.10 - 6.20, massif, 11 H, protons aromatiques + NH + NH₂
• 3.90, triplet mal résolu, 2 H, CH₂ en 6
• 3.00, triplet mal résolu, 2 H, CH₂ en 7
• Spectres infrarouge (KBr) (ν cm^-1, attribution) présentent tous les mêmes absorptions :
• 3458, 3371 cm^-1, NH; NH₂
• 3073 cm^-1, C=C-H
• 1638 cm^-1, C=N, C=C

The product thus obtained is light brown in color, with a yield of 17%.

• Mp = 228 ° C.
• Elementary analysis: C ₁₉ H ₁₅ N ₃ Cl ₂ , 1/2 H ₂ O Calculated% C: 62.48 H: 4.41 N: 11.50 Find % C: 62.29 H: 4.47 N: 11.43
• ¹ H NMR spectrum (CF ₃ COOD) (δ ppm, signal, n protons, attibution):
• 8.10 - 6.20, massive, 11 H, aromatic protons + NH + NH ₂
• 3.90, poorly resolved triplet, 2 H, CH ₂ in 6
• 3.00, poorly resolved triplet, 2 H, CH ₂ in 7
• Infrared spectra (KBr) (ν cm ^-1 , attribution) all have the same absorptions:
• 3458, 3371 cm ^-1 , NH; NH ₂
• 3073 cm ^-1 , C = CH
• 1638 cm ^-1 , C = N, C = C

b) 14-amino 6,7-dihydro chloride 12H-benzo- [f] indolo [2,3-a] quinolizinium (compound H).

Compound H is prepared according to a procedure identical to that described above in the context of the preparation of compound G but with the use of compound B instead of compound A.

• Pf supérieur à 260°C.
• Analyse élémentaire : C₁₉ H₁₆ N₃ Cl Calculé % C : 70,91 H : 5,01 N : 13,06 Trouvé % C : 70,33 H : 5,06 N : 12,71

The product obtained is mustard yellow in color with a yield of 56%.

• Mp above 260 ° C.
• Elementary analysis: C ₁₉ H ₁₆ N ₃ Cl Calculated% C: 70.91 H: 5.01 N: 13.06 Find % C: 70.33 H: 5.06 N: 12.71

c) 14-amino 6,7-dihydro chloride 12H-2-chloro benzo- [1] indolo [2,3-a] quinolizinium (compound I).

Compound I is prepared according to a procedure identical to that described above in the context of the preparation of compound G but with the use of compound C instead of compound A.

• Pf supérieur à 260°C.
• Analyse élémentaire : C₁₉ H₁₅ N₃ Cl₂, H₂O Calculé % C : 60,97 H : 4,38 N : 11,22 Trouvé % C : 61,32 H : 5,03 N : 10,52

The product obtained is light brown in color with a yield of 7%.

• Mp above 260 ° C.
• Elementary analysis: C ₁₉ H ₁₅ N ₃ Cl ₂ , H ₂ O Calculated% C: 60.97 H: 4.38 N: 11.22 Find % C: 61.32 H: 5.03 N: 10.52

d) 14-Amino 6,7-dihydro chloride 12H-3-chloro benzo- [1] indolo [2,3-a] quinolizinium (compound J).

Compound J is prepared according to a procedure identical to that described above in the context of the preparation of compound G but with the use of compound D instead of compound A.

• Pf supérieur à 260°C.
• Analyse élémentaire : C₁₉ H₁₅ N₃ Cl₂ Calculé % C : 64,06 H : 4,24 N : 11,79 Trouvé % C : 63,89 H : 4,48 N : 11,58

The product obtained is light brown in color with a yield of 52%.

• Mp above 260 ° C.
• Elementary analysis: C ₁₉ H ₁₅ N ₃ Cl ₂ Calculated% C: 64.06 H: 4.24 N: 11.79 Find % C: 63.89 H: 4.48 N: 11.58

e) 14-amino 6,7-dihydro chloride 12H-4-chloro benzo- [1] indolo [2,3-a] quinolizinium (compound K).

Compound K is prepared according to a procedure identical to that described above in the context of the preparation of compound G but with the use of compound E instead of compound A.

• Pf supérieur à 260°C.
• Analyse élémentaire : C₁₉H₁₅ N₃ Cl₂, H₂O Calculé % C : 60,97 H : 4,58 N : 11,22 Trouvé % C : 60,56 H : 4,66 N : 10,84

The product obtained is light brown in color with a yield of 6%.

• Mp above 260 ° C.
• Elementary analysis: C ₁₉ H ₁₅ N ₃ Cl ₂ , H ₂ O Calculated% C: 60.97 H: 4.58 N: 11.22 Find % C: 60.56 H: 4.66 N: 10.84

f) 14-amino 6,7-dihydro chloride 12H-3-bromo benzo- [1] indolo [2,3-a] quinolizinium (compound L).

Compound L is prepared according to a procedure identical to that described above in the context of the preparation of compound G but with the use of compound F instead of compound A.

• Pf supérieur à 260°C.
• Analyse élémentaire : C₁₉ H₁₅ N₃ Cl, Br, 2H₂O Calculé % C : 52,25 H : 4,38 N : 9,62 Trouvé % C : 52,28 H : 4,36 N : 9,66

The product obtained is mustard yellow in color with a yield of 12%.

• Mp above 260 ° C.
• Elementary analysis: C ₁₉ H ₁₅ N ₃ Cl, Br, 2H ₂ O Calculated% C: 52.25 H: 4.38 N: 9.62 Find % C: 52.28 H: 4.36 N: 9.66

IV- Study of the effects of the compounds of the invention on the CFTR: A) Methodology a) Culture of human epithelial cells and of recombinant cells CHO:

Several cell types are used for this study: the intestinal lines T84, Caco-2 and HT 29. Chinese-Hamster-Ovary cells (CHO-K1) were transfected using the vector pNUT (Tabcharani et al., 1991 ) incorporating or not (control cells) the normal or mutated CFTR cDNA (Chang et al., 1993). The cells are maintained in this specific medium and then seeded at low density on glass coverslips and cultured at 37 ° C. (5% CO ₂ ) before the patch-clamp experiments. The cell survival medium is composed of αMEM with fetal beef serum (7%) and antibiotics: antibiotics: 50 IU / ml penicillin and 50 µg / ml streptomycin and methotrexate (100 µM to 200 µM).

b) Principles of the molecular electrophysiology technique or patch clamp :

The electrical activity of cells is controlled by the presence and functioning of transmembrane pores, the ion channels. Ion channels are proteins whose conformational state can be modified in response to various factors: the transmembrane electric field, the binding of ligands or post-transcriptional biochemical reactions. The current passing through an ion channel is of the order of a billionth of an Ampere (pico Ampere, 1 pA = 10 ^-12 A). It can be measured by molecular electrophysiology techniques more commonly known as patch-clamp.

With conventional techniques for measuring transmembrane currents by intracellular microelectrodes, the thermodynamic background noise is at least a hundred times greater than the current flowing through a single ion channel. In under such conditions, the flow in a channel is masked by this noise, the variance increases with the mean current. Erwin Neher and Bert Sackman (see Hamill et al., 1981) of the Max Planck Institute in Gôttingen have shown that could by noise analysis estimate the current flowing in an ion channel. The thermodynamic background noise is proportional to the surface of the membrane. Thus, by limiting this, the background noise becomes lower than current flowing through the channel.

The patch-clamp technique results from the observation made by these two researchers and their collaborators: a glass micropipette applied to a membrane surface adheres to it so that the electrical resistance established between the pipette and the membrane reaches the value of gigaohm (1 Gohm = 10 ₉ ohm). Ohm's law gives the electrical resistance (R) with respect to the intensity of the current I (in Ampere) and the potential U (in Volt) imposed (equation 1) and makes it possible to determine the conductance (unit: the picoSiemens, ps) unit of channel g (equation 2). (equation 1) U = RI (equation 2) g = 1 / R

The interaction forces between the glass of the pipette and the phospholipids of the membrane make it possible to reduce the leakage currents, an essential step for the measurement of the current passing through an ion channel. The configuration thus obtained is designated by the term "cell-attached" or attached cell. By withdrawing the pipette from the attached cell configuration, a fragment (patch) of membrane is torn off which remains attached to the end of the pipette. The configuration thus obtained is called "inside-out" because the intracellular face of the membrane is found in the bath. The extracellular surface of the membrane is in contact with the solution contained in the pipette while the intracellular part is in contact with the perfusion medium of the experimental tank. An electronic assembly makes it possible to impose (to clamp) a potential difference (Vref-Vp) between the reference electrode of the bath (Vref) and the pipette (Vp) and to measure the resulting current I. If the ionic composition is the same on both sides of the membrane (symmetrical media), the intensity of the current I is then directly proportional to the potential difference imposed on the membrane. Points I (V) are generally distributed on a straight line whose slope and position define the unit conductance (g) of the channel and the current inversion potential, Erev, At inversion potential, the charge flow through the membrane is zero. The inversion potential will be defined by the Nernst equation (equation 3) where E represents the Nernst potential for the ion considered and C the concentration of the ion in the extracellular (Co) and intracellular (Ci) compartments. (equation 3) E = RT / z F Log Co / Ci

In cell-attached configuration, the potential of the electrode is added to that of the membrane which is around -60 mV. In all cells, the concentrations of potassium ions (K ⁺ ) and chlorine (Cl ^- ) are around 150 mM and 10 mM respectively. With a pipette containing 150 mM KCI, the Nernst potentials for the K ⁺ (E _K ) and Cl ^- (E _Cl ) ions will be close to zero and -50 mV, respectively. The inversion of the chlorine current will therefore be obtained in the vicinity of the resting potential, that is to say without applying a potential to the membrane (Vp = O mV). On the other hand, the reversal of a potassium current will be obtained by canceling the potential of the membrane, therefore by depolarizing it from 50 to 60 mV. This example illustrates the strategies used to assess the ionic nature of a channel. The information collected by this technique is represented by the fluctuation of an electric current (of the order of a millisecond, ms) which translates the transitions between the different conductance states of the channel.

c) Patch-clamp applied to the study of epithelial cells in culture.

Patch-clamp experiments are carried out on confluent cells. A fragment of the glass slide (cell support) is placed in an experimental tank (volume 600 µl or 1 ml) on the stage of an inverted microscope, equipped with phase contrast lighting (Olympus IMT2). The cell-attached and inside-out configurations are used (Hamill et al., 1981). The experiments are carried out at room temperature (20-22 ° C). The currents are amplified with a LIST EPC 7 amplifier (Darmstadt, Germany) (3 kHz filter) with a 2-5 kHz low pass filter (6-pole Bessel filter) and recorded with a DAT (Digital Audio Tape) after digitalization (16 bits) at 44 kHz. The data is then transferred to an Olivetti M28PC computer. The pipettes are made from 1 mm diameter glass tubes (Clark Electromedical Instrument) in two or three stages with a horizontal drawing machine (Flaming / Brown type, model P-87. Sutter Inst. Co. USA) . The pipettes filled with a 150 mM NaCl solution have a resistance of between 4 and 12MΩ. The potentials are expressed as the difference between the potential of the patch electrode and that of the bath. In the cell-attached configuration, they represent the change in potential with respect to the cell's resting potential. The junction potentials are evaluated by the potential of the electrode corresponding to a zero current (when the channels are closed). They are minimized by using an agar bridge establishing the connection between the bath and the reference electrode (earth) and containing the same solution as that of the pipette. The current inversion potential and the unit conductance of the channels are obtained from the current-voltage relationship (I / V) by linear regression. To determine the current-voltage relationship, the ion current amplitudes are measured from amplitude histograms. Amplitude histograms appear as the sum of two or more Gaussian distributions whose peaks correspond to the open and closed states of the channels present in the electrode. From these histograms we can determine: N, the total number of channels present in the patch; n, the number of simultaneously open channels (n = O, 1 2 ... N); Po, the probability of opening a channel; and I, the average current intensity in a channel. When the channels present are of the same type and supposed to open and close independently of each other, the probability of having n channels open simultaneously is given by the binomial distribution (equation 4) from which we deduce the individual probability Po (equation 5). (equation 4) P (n) = (N! / n! (Nn) Po not . (1-Po) nn (equation 5) Po = P (n) / N

The present study relates only to Cl ^- channels, an outgoing current should be interpreted as a movement of Cl ^- ions leaving the pipette towards the intracellular medium of the cells or towards the perfusion medium. The relative permeability P _X / P _Cl of an anion X ^- compared to Cl ions ^- was used to evaluate the ion selectivity of the channels in inside-out configuration. The Goldman-Hodgkin-Katz equation (equation 6) makes it possible to relate the ratio of permeabilities as a function of the inversion potential (Erev) obtained experimentally and the respective concentrations of the anions present. (equation 6) Erev = -RT / F In ([Cl - ] e + P X / P Cl [X - ] e / [Cl - ] i + P X / P Cl [X - ] i ) i and e: intracellular and extracellular ion concentration, R, T and F have their usual meaning.

For filling the patch electrodes, the composition of the saline solutions is (in mM); 150 NaCl, 2 MgCl ₂ , 10 TES (pH 7.4). The cell perfusion bath contains (in mM): 145 NaCl, 4 KCl, 2 MgCl ₂ , 0.5 CaCl ₂ , 10 TES (pH 7.4).

d) Measurement of short-circuit currents in the Ussing chamber.

The advantage of growing epithelia in chambers with a permeable bottom and in particular of digestive epithelia (line HT 29 and its different clones, line T84 or Caco2) has been widely demonstrated in cell biology studies. In this technique, the cells are cultured inside a cup where the bottom consists of a polystyrene membrane pierced with holes whose diameter (between 0.45 and 3 µm) and the distribution are carefully measured . It allows the attachment of cells without adding an additional matrix. It is transparent in a medium whose refractive index is close to that of water and allows the optical examination of the cell layer. However, there are limitations. Although the thinness of the membrane limits the retention of fluids, it cannot be excluded that it may constitute a trap for macromolecules or complexes such as gelosomes. This 5 cm ² cup is placed in a 6-well plate. Attachment and cell culture are carried out in the traditional way. A fortnight after seeding the cells form a tight monolayer. This tightness is proven by the appearance of electrical resistance or by the non-diffusion of macromolecules between the two mucous and serous compartments. The culture is stable for ten days while maintaining an identical culture medium in the two compartments. This culture of epithelia in chambers with a permeable bottom is quite useful for studying the nature and regulation of secretions and the passage of charged or uncharged molecules through the epithelium. The transepithelial transport will depend on the nature of the permeases present in the two apical or basolateral domains, on either side of the tight junction. The properties of the epithelium, which result in the passage of charged molecules, can be easily deduced from the measurement of the transepithelial potential or of the short-circuit current. When the molecules are neutral we use marked molecules to follow their movements.

e) Principles of the measurement method.

Schematically, transepithelial transport is the balance of cell transport and selective diffusion across the junction. Cell transport results from the activity of pairs of specific permeases located respectively on the apical pole and on the basal pole of the cell (Na ⁺ and Na ⁺ / K ⁺ ATPase channel, Cl ^- channel and Na ⁺ / K ⁺ / cotransporter Cl ^- , Na / glucose cotransporter and diffusional glucose transporter ...). When the transporter epithelium is sealed, the tight junction isolates two parts of the membrane which have a different potential compared to the medium. We can use a simple electrical analogy and consider them as circuit elements having respectively the potentials Vm (mucous) and Vs (serous). These two membranes in series therefore have a potential Vt which is the algebraic sum Vm + Vs. In the tissue used Vt can reach -5 mV under standard conditions. To determine the characteristics of the epithelium, three types of measurement can be used.

Open circuit measurement.

We measure the difference in potential existing on either side of the cell layer. Then at a fixed time, a current is sent into the circuit configurable i (µA) which causes a variation in potential difference ΔV proportional to the resistance of the circuit.

Short-circuit current measurement.

An adjustable current i is introduced into the circuit, which uses the resistance tissue to create a potential difference that will be added algebraically to the existing one. When the current has the value Isc (short-circuit current) Vt is no ; Isc x Rt - Et = 0 or Isc = Et / Rt. Under these conditions Vm-Vs = 0 and Vm = Vs, the two mucous and serous membranes have the same potential. To determine the resistance, a potential difference is imposed on the circuit. chosen and the resistance is measured by assessing the deviation of the current Isc which results.

Measurement in imposed voltage.

This is a special case of short-circuit current measurement where we requires the epithelium to have zero potential. Under these conditions we set the transepithelial potential without knowing the potential of each of the membranes. For this we superimpose a potential difference by extending the addition algebraic current which is used to measure resistance. We choose a time short for short-circuit current and a long time for super-addition. It is clear that the potential difference will depend, for a given resistance of the ability of the device to deliver a maximum current (100 µA; for 500 ohms the potential difference is 50 mV) but also to be able to measure the resulting potential difference (1 V).

To carry out these measurements, the cups are mounted in a Ussing changed. We use a "current-voltage clamp" control unit (WPI), coupled to a pulse generator allowing the production of curve intensity, voltage. The collected signal is digitized (MacLab) and processed using Chart software on an Apple Macintosh computer.

f) Properties of the epithelium tested.

First we will use the epithelium formed by the cells HT29. Adding glucose to the base medium (symmetrical saline medium, absence stimulator) in the mucous compartment causes an elevation of the transepithelial potential and short-circuit current without affecting the resistance. The epithelium functions as a glucose absorber which uses on the side mucosal a glucose dependent Na transporter, and presumably a diffusional transporter on the serous side. The transport of sodium associated with glucose causes a potential difference which can be inhibited by phlorizine while net glucose flux is measured with analogs of radiolabelled glucose placed in the mucous or serous compartments.

The addition of agents to the medium which cause an increase in the level of cAMP induces an increase in Vt and in Isc which is independent of glucose. It appears to be linked to the establishment of a transepithelial transport of Cl ^- (serous to mucous) and involves a basolateral chloride transporter on the serous side and a chloride channel on the mucous side. This transport of Cl ^- is associated with a transport of water of the same direction. The application, after an increase in the level of cAMP, of agents which cause a rise in the level of intracellular Ca ²⁺ leads to a new increase in Vt and Isc which associates, undoubtedly a passage of Ca ²⁺ mucous towards serous and a new passage from Cl ^- serous to mucous.

g) Measurement of radioactive tracer fluxes applied to the study of cells epithelial in culture.

This technique tracks the kinetics of iodide output. The cells are cultured in 12-well plates with dilution with 1/10 after passage. On day 3, the drugs to be tested are dissolved in medium B (37 ° C) depending on the desired concentration. Wells are washed 2 times with 1 ml of NaOH medium B, 0.1% glucose, which is then replaced by 1 ml of loading solution for 30 min.

The kinetics of iodide exit are carried out after eliminating the loading solution and washed the wells 3 times with 1.5 ml of medium B. For this 1 ml of medium B is left for 1 min in the well and recovered in a hemolysis tube to be replaced by 1 ml of new medium B. The first minute serves as control, the product to be tested is added from the second minute. The operation is repeated over 10 min then, those are removed with 1 ml of 0.1N NaOH 0.1% SDS. The content of each well is collected in a tube hemolysis after 25 min of stirring and the tubes are counted for 2 min in a gamma counter.

B) Results

The study concerns molecules of the benzoquinolizinium family, described above. They are tested for their ability to activate the CFTR channel. The screening of molecules as openers of the CFTR channel was carried out in measuring their effect on the efflux of radioactive iodide and on the chloride streams transmembrane (Becq et al., 1993a). These data have been supplemented by measuring the intracellular cyclic AMP (cAMP) rate and its variations in various experimental situations

. Three cellular models were used to assess the activation effect of CFTR by benzoquinoliziniums: the Xenopus oocyte injected with the RNA coding for CFTR (this RNA being designated hereinafter cRNA-CFTR); the recombinant CHO cell stably expressing the CFTR protein, and the human colonic cell of line HT29 constitutively expressing the CFTR protein. The CFTR channel being mainly regulated by proteins kinases A stimulated by the level of intracellular cAMP, the control experiments used cAMP derivatives capable of passing the membrane cell, and forskolin activator of the enzyme adenylate cyclase leading to the synthesis of cAMP in a cell. Figure 1 shows such activation obtained with 500 μM of cpt-cAMP (8- (4-chlorphenylthio) -adenosine-3 ', 5'-monophosphate, cyclic), a cAMP analog crossing the membrane of cells. Activation of the CFTR channel, measured by the iodide efflux, induces a increase in the amplitude of iodide efflux (expressed as a% of the content cell at time t = 0) and the output speed of iodide (slope of the curves originally).

1) ovocyte de Xénope : efflux de iodure radioactif et courant chlorure transmembranaire dans :

• ovocytes injectés avec l'ARNc-CFTR (notés CFTR sur la figure 1 B);
• les mêmes ovocytes en présence d'activateurs de la voie AMPc (cpt-AMPc, forskoline);
• ovocytes injectés avec de l'eau (notés "eau" sur la figure 1 B) aux lieu et place de l'ARNc-CFTR;
• les mêmes ovocytes mis en présence d'activateurs, ci-dessus mentionnés, de la voie AMPc.

2) cellule CHO: efflux de iodure radioactif dans

• cellules CHO non transfectées avec la protéine CFTR (notées, CHO-CFTR(-) sur la figure 2 B) en présence ou non d'activateurs, ci-dessus mentionnés;
• cellules CHO transfectées avec le gène CFTR (notées, CHO-CFTR(+) sur la figure 2 B) en présence ou non des activateurs, ci-dessus mentionnés.

3) cellule HT29 : efflux d'iodure radioactif dans

• cellules HT29 en l'absence d'activateur (notées basal sur la figure 3);
• cellules en présence d'activateurs (notées AMPc sur la figure 3), ci-dessus mentionnés.

The control experiments making it possible to evaluate the effectiveness of the molecules tested on the activity of the CFTR channel are the following:

1) Xenopus oocyte: efflux of radioactive iodide and current transmembrane chloride in:

• oocytes injected with cRNA-CFTR (denoted CFTR in FIG. 1B);
• the same oocytes in the presence of activators of the cAMP pathway (cpt-cAMP, forskolin);
• oocytes injected with water (noted "water" in Figure 1B) in place of the cRNA-CFTR;
• the same oocytes placed in the presence of activators, mentioned above, of the cAMP pathway.

2) CHO cell : efflux of radioactive iodide in

• CHO cells not transfected with the CFTR protein (noted, CHO-CFTR (-) in FIG. 2 B) in the presence or not of activators, mentioned above;
• CHO cells transfected with the CFTR gene (noted, CHO-CFTR (+) in FIG. 2 B) in the presence or not of the activators, mentioned above.

3) HT29 cell: efflux of radioactive iodide in

• HT29 cells in the absence of activator (noted basal in FIG. 3);
• cells in the presence of activators (denoted AMPc in FIG. 3), mentioned above.

The presence of a chloride channel activated by the increase in the level of intracellular calcium was tested in the presence of the calcium ionophore A23187. The effects of activators of cAMP, A23187 and benzoquinoliziniums were evaluated under these various experimental conditions (considered mutatis mutandis as control, noted basal) by their capacity to promote the efflux of radioactive iodide and to increase the current. transmembrane chloride.

Figures 2 A and 2 B show the activation of CFTR by 500 μM of cpt-AMPc in the CHO recombinant cell expressing CFTR. In the cell CHO (CFTR-) control cpt-AMPc has no effect. In cell HT29 the channel CFTR is stimulable by cAMP as shown by the increase in the amplitude of the iodide flow in the presence of 500 μM of cpt-AMPc (Figure 3 A and B).

Effect of benzoquinoliziniums on the activation of the CFTR channel in the CHO cell

Figure 4 shows the effect of the MPB-07 derivative (500µM) on the efflux iodide in the CHO (CFTR +) and CHO (CFTR-) cell. Activation of the efflux of iodide in the CHO cell (CFTR +) is comparable, in intensity and speed, at that induced by forskolin (5µM), activator of cAMP, in CHO cells (CFTR +) (Figure 5). The results concerning the effects of the derivative MPB-07 on the iodide efflux in CHO (CFTR-) and CHO (CFTR +) cells are summarized in the histograms of Figures 6 and 7, respectively. MPB-07 (500µM) stimulates iodide efflux as effectively as forskolin does (5µM) on CHO cells (CFTR +) (Figure 7). On these same cells, A23187 (10µM) has no effect. In CHO (CFTR-) cells, forskolin (5µM) A23187 (10µM), either separately or added together, as well as MPB-07 (500µM) do not significantly modify the basal efflux level iodide (Figure 6).

Effect of benzoquinoliziniums on the activation of the CFTR channel in the HT29 cell

The effect of MPB-07 on the efflux of iodide in the recombinant cell CHO is reproduced in the HT29 epithelial cell. The application of 500µM of cpt-AMPc (figure 8 A) or 500µM of MPB-07 (figure 8 B) triggers, with a similar speed and amplitude an increased iodide efflux compared to basal level (without activator) (Figure 9).

Effect of benzoquinoliziniums on the activation of the expressed CFTR channel in the Xenopus oocyte

MPB-07 (500µM) stimulates the efflux of iodide in the Xenopus oocyte. This activation (Figure 10 A) is comparable to that obtained by the application of 500 µM cpt-cAMP (Figure 10 B) and significantly different (Figure 11) from the efflux measured in the stimulated uninjected oocyte (cAMP, and MPB-07) and not stimulated (basal) (Figure 11, water) and in the unstimulated but expressing oocyte CFTR (Figure 10 A, efflux noted CFTR basal).

Structure-function study of benzoquinoliziniums and correlation with the opening of the CFTR

The study focused on 16 benzoquinolizinium nucleus derivatives.

Tables 1 and 2 show the chemical structure of the compounds of family of benzoquinoliziniums tested here as activator of the CFTR channel.

Tables 1 and 2 present the results relating to the efflux measured in the presence of the various compounds tested on the recombinant CHO (CFTR +) cell. The base compound, phenanthrene (Table 2) does not activate the CFTR channel. Two series of molecules were tested: NH ₂ series (table 1, MPB-26, MPB-01 to 04; table 2: MPB-24) and OH series (table 1, MPB-05 to 08, MPB-27, 29 , 30 and 32; Table 2: MPB-25). The activation percentages of the CFTR channel are given in the corresponding tables. They show that the OH series activates CFTR with percentages between 15 and 110%. The NH ₂ series is less active (10 to 30%).

In the OH series, the effectiveness is as follows: MPB-05, 08, 25, 32 <30 <29 <06 <27, 07

• 9 composés avec OH en position 6 : 45 % d'activation du canal CFTR
• 6 composés avec NH₂ en position 6 : 13 % d'activation du canal CFTR.

From all of the compounds studied, it appears that the presence of the OH group in position 6 is decisive as regards the capacity to activate the CFTR channel:

• 9 compounds with OH in position 6: 45% activation of the CFTR channel
• 6 compounds with NH ₂ in position 6: 13% activation of the CFTR channel.

Effect of benzoquinoliziniums on intracellular cAMP

FIG. 12 shows the levels of cellular cAMP in the CHO cell recombinant measured after 5 min in the presence of 5 μM of forskolin (activator cAMP synthesis enzyme; adenylate cyclase), 10 µM rolipram (an inhibitor of the cAMP degradation enzyme: phosphodiesterase from type IV) and 500 µM of MPB-07. The same level of cAMP is reached in presence of compounds MPB-07 and rolipram but only MPB-07 triggers activation of the CFTR channel. The forskolin effect on cAMP is multiplied by a factor of about 4 suggesting that this effect is purely dependent on cAMP. Rolipram has no CFTR activating effect measured in iodide flow and patch clamp. These results show that the compound MPB-07 stimulates the channel CFTR by a pathway independent of the cellular cAMP pathway.

C) Conclusion

These results show that the compound MPB-07 and certain members of the family of benzoquinoliziniums stimulate the opening of the CFTR channel by a independent of cAMP or intracellular calcium. These molecules therefore represent a new family of CFTR channel activators.

Legends of figures

• Figure 1 : effect of cpt-cAMP on the efflux of radioactive iodide in the Xenopus oocyte;
  • Figure 1 A : ¹²⁵ I efflux curves (% on the ordinate) as a function of time (min. on the abscissa); the curve passing through points represented by black squares corresponds to the efflux of ¹²⁵ I measured as a function of time in oocytes injected with the cRNA-CFTR (curve designated CFTR basal); the curve passing through points represented by white squares corresponds to the efflux of ¹²⁵ I measured as a function of time in oocytes injected with the cRNA-CFTR and activated by cpt-cAMP (curve designated CFTR + cAMP);
  • FIG . 1B: histograms of the ¹²⁵ I efflux in the oocytes not activated by cpt-cAMP (represented in black) and in the oocytes activated by cpt-cAMP (represented in white); on the left are represented the oocytes activated or not by cpt-cAMP and injected with water (noted "water"); on the right are represented the oocytes activated or not by cpt-AMPc and injected with cRNA-CFTR (noted "CFTR"); n represents the number of experiments.
• Figure 2: Effect of cpt-cAMP on the efflux of radioactive iodide in CHO cells;
  • Figure 2 A : ¹²⁵ I efflux curves (% on the ordinate) as a function of time (min. on the abscissa); the curve passing through points represented by black triangles corresponds to the efflux of ¹²⁵ I measured as a function of time in CHO cells not activated by cpt-cAMP (curve designated basal); the curve passing through points represented by white triangles corresponds to the efflux of ¹²⁵ I measured as a function of time in CHO cells activated by cpt-cAMP (curve designated cAMP);
  • FIG. 2B : histograms of the ¹²⁵ I efflux in CHO cells not activated by cpt-cAMP (shown in white); on the left are represented the CHO cells activated or not by cpt-AMPc and not transfected with the CFTR gene (noted CHO (CFTR-)); on the right are represented the CHO cells activated or not by cpt-AMPc and transfected with the CFTR gene (noted CHO (CFTR +)); n represents the number of experiments.
• Figure 3 : Effect of cpt-cAMP on the efflux of radioactive iodide in HT29 cells;
  • Figure 3 A : efflux curves of ¹²⁵ I (% on the ordinate) as a function of time (min. on the abscissa); the curve passing through points represented by black squares corresponds to the efflux of ¹²⁵ I measured as a function of time in HT29 cells not activated by cpt-cAMP (curve designated basal); the curve passing through points represented by white squares corresponds to the efflux of ¹²⁵ I measured as a function of time in HT29 cells activated by cpt-cAMP (curve designated cAMP);
  • FIG. 3B : histograms of the ¹²⁵ I efflux in HT29 cells not activated by cpt-AMPc (in black), and in HT29 cells activated by cpt-AMPc (in white).
• Figure 4 : effect of the MPB-07 derivative (500µM) on the efflux of ¹²⁵ I (% on the ordinate) as a function of time (min on the abscissa) in the CHO cell; the curve passing through points represented by white circles corresponds to the measurement of the efflux of ¹²⁵ I as a function of time in CHO cells activated by MPB-07 and transfected with the CFTR gene (curve designated MPB-07 (CFTR +) the curve passing through points represented by black circles corresponds to the measurement of the 125I efflux as a function of time in CHO cells activated by MPB-07 but not transfected with the CFTR gene (curve designated MPB-07 (CFTR- )).
• Figure 5 : effect of "forskolin" (500 µM) on the efflux of ¹²⁵ I (% on the ordinate) as a function of time (min on the abscissa) in the CHO cell; the curve passing through points represented by white triangles corresponds to the measurement of the efflux of ¹²⁵ I as a function of time in CHO cells activated by forskolin and transfected with the CFTR gene (curve designated forskolin CHO (CFTR +)); the curve passing through points represented by black triangles corresponds to the measurement of the ¹²⁵ I efflux as a function of time in CHO cells not activated by forskolin and transfected with the CFTR gene (curve designated basal CHO (CFTR +)).
• Figure 6: Histograms of the ¹²⁵ I efflux in CHO cells not transfected with the CFTR gene (designated CHO (CFTR-)) and:
  • not activated (basal)
  • activated by forskolin (forskolin)
  • activated by A23187 (A23187)
  • activated by A23187 and forskolin (A23187 + fsk)
  • activated by MPB-07 (MPB-07)
    n represents the number of experiments.
• Figure 7 : Histograms of the ¹²⁵ I efflux in CHO cells transfected with the CFTR gene (designated CHO (CFTR +)) and:
  • not activated (basal)
  • activated by forskolin (forskolin)
  • activated by MPB-07
  • activated by A23187 (A23187)
    n represents the number of experiments.
• Figure 8 : compared effects of cpt-cAMP (500 µM) and MPB-07 (500 µ M) on the efflux of ¹²⁵ I (% on the ordinate) as a function of time (min on the abscissa) in HT29 cells;
  • FIG. 8 A : the curve passing through points represented by black squares corresponds to the measurement of the efflux of ¹²⁵ I as a function of time in non-activated HT29 cells (curve designated basal); the curve passing through points represented by white squares corresponds to the measurement of the efflux of ¹²⁵ I as a function of time in HT29 cells activated by cpt-cAMP (curve designated cAMP);
  • FIG. 8B : the curve passing through points represented by white squares corresponds to the measurement of ¹²⁵ I as a function of time in non-activated HT29 cells (curve designated basal); the curve passing through points represented by black squares corresponds to the measurement of the efflux of ¹²⁵ I as a function of time in HT29 cells activated by MPB-07 (curve designated MPB-07).
• Figure 9 : Histogram of the ¹²⁵ I efflux in non-activated HT29 cells (basal), activated by cpt-AMPc (AMPc) and activated by MPB-07 (MPB-07); n represents the number of experiments.
• Figure 10 : Compared effects of cpt-cAMP (500µM) on the efflux of ¹²⁵ I (% on the ordinate) as a function of time (not on the abscissa) in Xenopus oocytes:
  • FIG. 10 A : the curve passing through points represented by white circles corresponds to the measurement of the efflux of ¹²⁵ I as a function of time in the oocytes injected with water (curve designated basal water); the curve passing through points represented by black squares corresponds to the measurement of the efflux of ¹²⁵ I as a function of time in the oocytes injected with the cRNA-CFTR (curve designated basal CFTR); the curve passing through points represented by white squares corresponds to the measurement of the efflux of ¹²⁵ I as a function of time in the oocytes injected with the cRNA-CFTR and activated with cpt-cAMP (curve designated CFTR + cAMP);
  • FIG. 10B : the curve passing through points represented by white squares corresponds to the measurement of the efflux of ¹²⁵ I as a function of time in the oocytes injected with the cRNA-CFTR and activated by MPB-07 (curve designated MPB -07).
• Figure 11 : Histograms of the ¹²⁵ I efflux in Xenopus oocytes not activated (basal), or activated by cpt-AMPc (AMPc), or activated by MPB-07 (MPB-07), these oocytes being either injected with water (labeled "water" on the left), or injected with cRNA-CFTR (labeled "CFTR" on the right); n represents the number of experiments.
• Figure 12 : Histograms representing cAMP levels (measured in nmol per mg of protein) in CHO cells transfected with the CFTR gene and not activated (basal), or activated by forskolin (forskolin), or activated by rolipran (rolipran ), or activated by MPB-07 (MPB-07).
  
  

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Claims (16)

1. Use of benzo[c]quinolizinium derivatives of general formula (III) which follows:

   

      in which:

   heterocycle A is aromatic or non-aromatic, it being understood that in the latter case the nitrogen atom of this heterocycle is linked by a double bond to the carbon in position 4a,

   R₁, R₂, R₃, R₄, R₅, R₇, R₈, R₉ and R₁₀, represent, independently of each other:

   a hydrogen, or bromine or fluorine atom, or

   a halogen atom, in particular a chlorine atom, or

   an alkyl, alkoxy, carbonyl or oxycarbonyl group, linear or branched, with 1 to 10 carbon atoms, if appropriate these groups being substituted, in particular by a halogen, and/or by a hydroxyl, and/or by an amine (primary, secondary or tertiary), and/or by an aromatic and/or aliphatic cycle, with 5 to 10 carbon atoms in the cycle, these cycles being themselves, if appropriate, substituted in particular by a halogen, and/or by a hydroxyl, and/or by an amine (primary, secondary or tertiary), and/or by an alkyl, alkoxy, carbonyl or oxycarbonyl group, these groups being as defined above, or

   an aromatic or aliphatic cycle, with 5 to 10 carbon atoms in the cycle, this cycle being itself, if appropriate, substituted in particular by a halogen, and/or by a hydroxyl, and/or by an amine (primary, secondary or tertiary), and/or by an alkyl, alkoxy, carbonyl or oxycarbonyl group, these groups being as defined above, or

   an -OR_a group, R_a representing a hydrogen atom or an alkyl, carbonyl or oxycarbonyl group, linear or branched, these groups being as defined above, or an aromatic or aliphatic cycle, these cycles being as defined above, or

   when R₁ and R₂, or R₃ and R₄, and/or R₄ and R₅, and/or R₇ and R₈, and/or R₈ and R₉, and/or R₉ and R₁₀, do not represent the different atoms or groups or cycles mentioned above, then R₁ in combination with R₂, or R₂ in combination with R₃, and/or R₃ in combination with R₄, and/or R₄ in combination with R₅, and/or R₇ in combination with R₈, and/or R₈ in combination with R₉, and/or R₉ in combination with R₁₀, form respectively with C₁ and C₂, or with C₂ and C₃, or with C₃ and C₄, or with C₄, C_4a and C₅, or with C₇ and C₈, or with C₈ and C₉, or with C₉ and C₁₀, an aromatic or aliphatic cycle, with 5 to 10 carbon atoms, if appropriate this cycle being substituted, in particular by a halogen, and/or by an alkyl, alkoxy, carbonyl or oxycarbonyl group, and/or an aromatic or aliphatic cycle, these groups or cycles being as defined above, or

   when R₃ and R₄ do not represent the different atoms or groups or cycles mentioned above, then R₃ in combination with R₄ form an indole group of formula

   

      in which R_a is as defined above,

   Y represents:

   an -OR_d group, R_d representing a hydrogen atom or an alkyl, carbonyl or oxycarbonyl group, linear or branched, these groups being as defined above, or an aromatic or aliphatic cycle, these cycles being as defined above, or

   an NR_eR_f group, R_e and R_f independently of each other, representing an alkyl, alkoxy, carbonyl or oxycarbonyl group, linear or branched, these groups being as defined above, or an aromatic or aliphatic cycle, these cycles being as defined above,

   it being understood that when R_d, or at least one of R_e and R_f do not represent one of the different atoms or groups or cycles mentioned above, then R_d, or at least one of R_e and R_f, in combination with R₅, or in combination with R₇, form respectively with C₅ or C₆, or with C₆, C_6a or C₇, an aromatic or aliphatic heterocycle with 5 to 10 carbon atoms, if appropriate substituted, in particular by a halogen, and/or an alkyl, alkoxy, carbonyl or oxycarbonyl group, and/or an aromatic or aliphatic cycle, these groups or cycles being as defined above,

   X represents an atom in anionic form, such as a halogen atom, in particular a bromine or chlorine atom, or a group of atoms in anionic form, such as a perchlorate, and the nitrogen of heterocycle A of formula (I) is in quaternary form and is linked on the one hand by a covalent bond to the carbon in position 11, and, on the other hand, by an ionic bond to X defined above,

   it being understood that when R₁ and R₁₀ do not represent one of the different atoms or groups or cycles mentioned above, then R₁ in combination with R₁₀ form with C₁, the nitrogen of heterocycle A of formula (I), C₁₁, and C₁₀, an aromatic or aliphatic heterocycle with 5 to 10 carbon atoms, if appropriate substituted, in particular by a halogen, and/or an alkyl, alkoxy, carbonyl or oxycarbonyl group, and/or by an aromatic or aliphatic cycle, these groups or cycles being as defined above,

      for the preparation of medicaments intended for the treatment of pathologies, in particular pulmonary, digestive or cardiac, linked to transmembrane ionic flux disorders, in particular chlorine and, if appropriate, bicarbonate, in the organism (human or animal), in particular for the preparation of medicaments intended for the treatment of mucoviscidosis, or for the prevention of rejection of cytotoxic drugs (in particular antitumoral), for the treatment of obstructions to the bronchial routes or digestive tracts (in particular pancreatic or intestinal).
2. Use according to claim 1 of derivatives of general formula (IIIa) which follows:

   

      in which:

   R₁ and R₂ represent a hydrogen atom, or form in combination with C₁ and C₂ an aromatic cycle with 6 carbon atoms,

   Y represents an -OH or -NH₂ or -NHCOCH₃ group,

   R₇, R₈, R₉ and R₁₀, represent a hydrogen atom or one of R₇, R₈, R₉ and R₁₀ represents a halogen atom, in particular a chlorine, bromine or fluorine atom,

   X represents a halogen atom in anionic form, in particular a bromine atom Br^-, or a chlorine atom Cl^-, or a group of atoms in anionic form, in particular a perchlorate ClO₄ ^-.
3. Use according to claim 1 or to claim 2, of compounds of formula (IIIa) in which:

   R₁ and R₂ represent a hydrogen atom,

   Y represents an -OH group,

   X represents a halogen atom in anionic form, in particular a bromine atom Br, or a chlorine atom Cl^-, or a group of atoms in anionic form, in particular a perchlorate ClO₄ ^-,

   R₇, R₈, R₉ and R₁₀, represent independently of each other a hydrogen atom or a halogen atom, in particular a chlorine, bromine or fluorine atom.
4. Use according to one of claims 1 to 3, of benzo[c]quinolizinium derivatives of general formula (IIIa) chosen from the following:

   

   

   

   

   

   

   

   

   

   

   

   

   

   

   

   

   
5. Use according to claim 1 of derivatives of general formula (IIIb) which follows:

   

      in which:

   R_a represents a hydrogen atom,

   R₁ and R₂ represent a hydrogen atom, and there is no double bond between the two carbons carrying R₁ and R_2,

   R₅ represents a hydrogen atom,

   R₇, R₈, R₉, and R₁₀ represent a hydrogen atom, or one of R₇, R₈, R₉, and R₁₀ represents a halogen atom, in particular a chlorine, bromine or fluorine atom,

   Y represents -NH₂,

   X represents a halogen atom in anionic form, in particular a bromine atom Br^-, or a chlorine atom Cl^-, or a group of atoms in anionic form, in particular a perchlorate ClO₄ ^-.
6. Use according to claim 1, of derivatives of general formula (IIIb) chosen from the following:

   

   compound G: R₇ = Cl, R₈ = R₉ = R₁₀ = H,

   compound H: R₇ = R₈ = R₉ = R₁₀ = H,

   compound I: R₈ = Cl, R₇ = R₉ = R₁₀ = H,

   compound J: R₉ = Cl, R₇ = R₈ = K₁₀= H,

   compound K: R₁₀ = Cl, R₇ = R₈ = R₉ = H,

   compound L: R₉ = Br, R₇ = R₈ = R₁₀ = H.
7. Pharmaceutical compositions characterized in that they contain, as active ingredient(s), at least one of the derivatives of general formula (III) as defined in one of claims 1 to 6, in association with a physiologically acceptable vehicle.
8. Pharmaceutical compositions according to claim 7, characterized in that they are presented in a form which can be administered by oral route, in particular in the form of tablets or capsules, or in a form which can be administered by parenteral route, in particular in the form of preparations which are injectable by intravenous, intramuscular or sub-cutaneous route, or also via the airways, in particular by pulmonary route in the form of aerosols.
9. Pharmaceutical compositions according to claim 7 or to claim 8, characterized in that the quantities of active ingredient(s) are such that the daily dose of active ingredient(s) is approximately 0.1 mg/kg to 5 mg/kg, in particular approximately 3 mg/kg, in one or more doses.
10. Benzo[c]quinolizinium derivatives of formula (III) which follows:

    

    in which R₁, R₂, R₃, R₄ R₅, R₇, R₈, R₉, R₁₀, X and Y are as defined in claim 1, the compounds of the following formula being excluded:

    

    

    

    

    

    
11. Derivatives according to claim 10, of general formula (IIIa) which follows:

    

       in which:

    R₁ and R₂ represent a hydrogen atom, or form in combination with C₁ and C₂ an aromatic cycle with 6 carbon atoms,

    Y represents an -OH or -NH₂ or NHCOCH₃ group,

    R₇, R₈, R₉ and R₁₀, represent a hydrogen atom or one of R₇, R₈, R₉ or R₁₀ represents a halogen atom, in particular a chlorine, bromine or fluorine atom,

    X represents a halogen atom in anionic form, in particular a bromine atom Br^-, or a chlorine atom Cl^-, or a group of atoms in anionic form, in particular a perchlorate ClO₄ ^-.
12. Derivatives according to claim 11, of formula (IIIa) in which:

    R₁ and R₂ represent a hydrogen atom,

    Y represents an -OH group,

    X represents a halogen atom in anionic form, in particular a bromine atom Br^-, or a chlorine atom Cl^-, or a group of atoms in anionic form, in particular a perchlorate ClO₄ ^-,

    R₇, R₈, R₉ and R₁₀, represent independently of each other a hydrogen atom or a halogen atom, in particular a chlorine, bromine or fluorine atom.
13. Benzo[c]quinolizinium derivatives according to one of claims 10 to 12, of general formula (IIIa) chosen from the following:

    

    7

    

    

    

    

    

    

    

    

    

    

    
14. Derivatives according to claim 10, of general formula (IIIb) which follows:

    

       in which R_a, R₁, R₂, R₅, R₇, R₈, R₉, R₁₀, X and Y are as defined in claim 1, and in particular the compounds of formula (IIIb) in which:

    R_a represents a hydrogen atom,

    R₁ and R₂ represent a hydrogen atom, and there is no double bond between the two carbons carrying R₁ and R₂,

    R₅ represents a hydrogen atom,

    R₇, R₈, R₉, and R₁₀ represent a hydrogen atom, or one of R₇, R₈, R₉, or R₁₀ represents a halogen atom, in particular a chlorine, bromine or fluorine atom,

    Y represents -NH₂,

    X represents a halogen atom in anionic form, in particular a bromine atom Br^-, or a chlorine atom Cl^-, or a group of atoms in anionic form, in particular a perchlorate ClO₄ ^-.
15. Derivatives according to claim 14, of general formula (IIIb) chosen from the following:

    

    compound G: R₇ = Cl, R₈ = R₉ = R₁₀ = H,

    compound H: R₇ = R₈ = R₉ = R₁₀ = H,

    compound I: R₈ = Cl, R₇ = R₉ = R₁₀ = H,

    compound J: R₉ = Cl, R₇ = R₈ = R₁₀ = H,

    compound K: R₁₀ = Cl, R₇ = R₈ = R₉ = H,

    compound L: R₉ = Br, R₇ = R₈ = R₁₀ = H.
16. Preparation process for derivatives as defined in claim 1, characterized in that it includes the following steps:

    treatment of the derivative of formula (A) in which R₁, R₂, R₃, R₄ and R₅ are as defined in claim 1, with phenyllithium or lithium diisopropyl amide, advantageously in ether or THF, which leads to the obtaining of derivatives of formula (B) in which R₁, R₂, R₃, R₄ and R₅ are as defined in claim 1, according to the following reaction diagram:

    

    condensation of the derivative of formula (B) obtained in the previous stage with the derivative of formula (C) in which R₇, R₈, R₉, R₁₀ and X are as defined in formula (I), which leads to the obtaining of derivatives of formula (D) in which R₁, R₂, R₃, R₄, R₅, R₇, R₈, R₉, R₁₀ and X are as defined in claim 1, according to the following reaction diagram:

    

    treatment of the compound of formula (D) by the addition of H₂O, which leads to the obtaining of the following derivative of formula (II), in which R₁ to R₅, R₇ to R₁₀, and X are as defined in claim 1, and Y represents -NH₂,

    

    if appropriate, treatment of the above-mentioned compound of formula (II), with a derivative containing the R_e and R_f groups as defined in claim 1, this derivative being capable of reacting with the nitrogen atom linked to the carbon in position 6 of the above-mentioned compound of formula (II), in particular by a halide of R_e and/or R_f while having, if necessary, taken care to protect beforehand those other functions present on the above-mentioned compound of formula (II) and capable of reacting with the derivative containing the above-mentioned R_e and R_f groups, which leads to the obtaining of the following compound of formula (II) in which R, to R₅, R₇ to R₁₀ and X are as defined above and Y represents an -NR_eR_f group as defined in claim 1,

    

    if appropriate, hydrolysis, in particular by the action of sulphuric acid (pH3) at 40°C, of the above-mentioned compound of formula (II) in which Y represents NH₂, which leads to the obtaining of the following compound of formula (II), in which R₁ to R₅, R₇ to R₁₀ and X are as defined in claim 1, and Y represents an -OH group,

    

    if appropriate, treatment of the above-mentioned compound of formula (II), in which Y represents an -OH group, with a derivative containing the R_d group, as defined in claim 1, this derivative being capable of reacting with the oxygen atom linked to the carbon in position 7 of the above-mentioned compound of formula (II), in particular by a halide of R_d, while having, if necessary, taken care to protect beforehand those other functions present on the above-mentioned compound of formula (II) and capable of reacting with the derivative containing the above-mentioned R_d group, which leads to the obtaining of the following compound of formula (II) in which R₁ to R₅, R₇ to R₁₀ and X are as defined above and Y represents an -OR_d group as defined in claim 1,

    

    heating, advantageously at 200°C, the above-mentioned compounds of formula (II) in which Y represents -NH₂ or -OH, which leads respectively to the following compounds of formula (III), in which R₁ to R₅, R₇ to R₁₀ and X are as defined in claim 1 and Y represents an -NH₂ or -OH group,

    

    

    if appropriate, treatment of the above-mentioned compound of formula (III), in which Y represents an -NH₂ group, with a derivative containing the R_e and R_f groups as defined in claim 1, this derivative being capable of reacting with the nitrogen atom linked to the carbon in position 6 of the above-mentioned compound of formula (III), in particular by a halide of R_e and/or R_f while having, if necessary, taken care to protect beforehand those other functions present on the above-mentioned compound of formula (III) and capable of reacting with the derivative containing the above-mentioned R_e and R_f groups, which leads to the obtaining of the following compound of formula (III) ii which R₁ to R₅, R₇ to R₁₀ and X are as defined above and Y represents an -NR_eR_f group as defined in claim 1,

    if appropriate, treatment of the above-mentioned compound of formula (III), in which Y represents an -OH group, with a derivative containing the R_d group, as defined in claim 1, this derivative being capable of reacting with the oxygen atom linked to the carbon in position 7 of the above-mentioned compound of formula (III), in particular by a halide of R_d, while having, if necessary, taken care to protect beforehand those other functions present on the above-mentioned compound of formula (III) and capable of reacting with the derivative containing the above-mentioned R_d group, which leads to the obtaining of the following compound of formula (III) in which R₁ to R₅, R₇ to R₁₀ and X are as defined above and Y represents an -OR_d group as defined in claim 1,

    

    if appropriate, hydrogenation of the above-mentioned compounds of formulae (II) or (III), in particular by catalytic hydrogenation in the presence of platinum oxide at reduced pressure, which leads to the obtaining of the following compounds of formulae (II) or (III):

    

    

       in which R₁ to R₅, R₇ to R₁₀, X and Y are as defined above.

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